IntroductionHuman adenoviruses are responsible for a variety ofillnesses. In children, adenoviruses account for 5–10per cent of respiratory illnesses including commoncold, nasopharyngitis, pharyngitis, tonsillitis, laryn-gotracheitis, bronchiolitis, and pneumonia. Thereare also other manifestations, such as conjunctivitis,gastroenteritis, and neurological symptoms. The

clinical picture of adenoviral infection is character-ized by high grade and prolonged fever.1,2

Adenoviral infection can be diagnosed specificallyby virus isolation. Types and subtypes of aden-oviruses can be determined on immunofluorescencetest, neutralization test (NT), subgenus polymerasechain reaction (PCR), PCR-restriction fragmentlength polymorphism (RFLP), and RFLP. Thesemethods provide essential tools for epidemiologicalstudies.3 Most laboratories do not serotype aden-ovirus isolates, which makes it more difficult toidentify outbreaks of infection caused by a specifictype. We have noted that adenoviral infectionsincreased in southern Taiwan, which were detectedat the Kaohsiung Medical University Hospital(KMUH) Virology Laboratory during the last fouryears, 1999–2002. Adenovirus type 3 and type 7 werethe major types between November 1999 and March2000, while type 4 was found mainly in October 2000and September 2001. Adenovirus type 7 and type 4emerged in 1999 and 2000 and declined in 2001 and

Journal of Tropical Pediatrics, Vol. 50, No. 5 Oxford University Press 2004; all rights reserved 279

Respiratory Adenoviral Infections in Children: A Study ofHospitalized Cases in Southern Taiwan in 2001–2002

by Hsiu-Lin Chen,a Shyh-Shin Chiou,a Hui-Pin Hsiao,a Guan-Ming Ke,b Yung-Cheng Lin,b Kuei-Hsiang Lin,b and Yuh-JyhJongc

aDepartment of Pediatrics, Kaohsiung Municipal Hsiaokang Hospital, Kaohsiung, TaiwanbDepartment of Clinical Laboratory, Kaohsiung Medical University, Kaohsiung, TaiwancDepartment of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan

SummaryAdenoviruses account for 5–10 per cent of respiratory illnesses in children. To analyse the clinicalfeatures and the temporal frequency in acute adenoviral respiratory infections in hospitalizedchildren in southern Taiwan, a total of 4333 children who were admitted to the Department of Pedi-atrics, Kaohsiung Municipal Hsiaokang (KMHK) Hospital, with clinical evidences of acute respi-ratory infections between January 2001 and December 2002 were studied. Adenoviruses wereisolated from 317 patients with an isolation rate of 7.67 per cent. Serotype analysis was performedby polymerase chain reaction (PCR) and/or PCR-restriction fragment length polymorphism (PCR-RFLP) in 186 specimens. In 2001, adenovirus type 4 was found in the majority (57 per cent),followed by type 1.5.6 (15 per cent), type 2 (13 per cent), type 14 (8 per cent), type 3 (5 per cent),and type 7 (2 per cent). In 2002, type 3 became the major type (46 per cent), whereas the previ-ously predominant type 4 decreased to 6 per cent, and type 7 increased from 2 to 19 per cent. Thesymptoms and signs included fever (98.7 per cent), cough (77.6 per cent), abnormal breathingsounds (crackles and/or wheezing 23.3 per cent), abdominal pain (18.9 per cent), vomiting (21.8 percent), and diarrhea (25.2 per cent). The mean duration of fever was 4.8 days (range 0–19 days). Inthe 186 cases in whom serotypes were analysed, pharyngitis and tonsillitis (47.8 per cent) were themost common presentation, followed by pneumonia (25.2 per cent), bronchitis (12.9 per cent), andpharyngoconjunctival fever (PCF) (7.6 per cent). Children between 4 and 8 years old were the mostcommon group of patients with respiratory adenoviral infections. Our patients all had good prog-nosis. This adenoviruses molecular epidemiological study provides information that helpsphysicians in clinical differential diagnosis and treatment of respiratory adenoviral infection inchildren in southern Taiwan.

AcknowledgementsWe thank the staff of the KMUH Virology Laboratory whosediligent and expert diagnostic work allowed the identification ofadenoviruses utilized in this study. We also thank Dr Wu-Yuan Chenfor his critical review of this manuscript and Miss Lih-Ru Fang forher diligent secretarial work.

Correspondence: Hsiu-Lin Chen, Department of Pediatrics,Kaohsiung Municipal Hsiaokang Hospital No. 482, Shan-MingRd., Hsiao-Kang Dist., Kaohsiung 812, Taiwan. E-mail<k84062@kmhk.kmu.edu.tw>.

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2002, respectively.4 To provide more informationabout the clinical features and the temporalfrequency in acute adenoviral respiratory infectionsdue to different adenoviral serotype in hospitalizedchildren in southern Taiwan, isolates collected fromKaohsiung Municipal Hsiaokang (KMHK) hospitalduring 2001–2002 were studied using PCR, PCR-RFLP, and/or RFLP.

Materials and Methods

PatientsThe study population included 4333 children whowere admitted to the Department of Pediatrics,KMHK Hospital, with clinical evidence of acuterespiratory infections between January 2001 andDecember 2002. The KMHK Hospital is a localteaching general hospital located in southern Kaoh-siung with about 200–230 pediatric patient admis-sions per month. The majority of our patients comefrom southern Taiwan. Throat swabs were collectedat admission. The samples were sent to KMUHlaboratory for bacterial culture, virus isolation, andidentification. Blood examinations including whiteblood cell counts, biochemistry, and C-reactiveprotein were made. Adenoviral diagnosis was madeby throat swab viral isolation. Medical records ofpatients with respiratory adenoviral infections werereviewed for clinical symptoms, signs, laboratorydata, courses of treatment, and prognosis.

Virus isolation and identificationThroat swabs collected from patients were culturedin HeLa and/or A459 cells and identified using Adspecific monoclonal antibody by immunofluorescentassay from which adenvirus specific cytopathiceffect was recognized. Virus stocks were prepared inHeLa cell with Eagle’s minimum essential mediumsupplemented with 2 per cent fetal calf serum andantibiotics.

DNA extractionFor PCR-RFLP, viral DNA was extracted using ablood purification kit (Amershan Pharmacia BiotechInc., NJ) according to the manufacturer’s instruc-tions.

Adenovirus typing by PCR-RFLPAdenoviruses were typed by PCR-RFLP asdescribed in previous reports.5,6 The primer pairAdnU-S� (5�-TTCCCCATGGCNCACAACAC-3;20743–20762) and AdnU-A (5�-GCCTCGAT-GACGCCG CGGTG-3�; 21679–21698) were used toamplify a 956 bp product in the hexon region.5,6 PCRproducts were subjected to digestion with threerestriction enzymes, Sty I (EcoT 141), Hae III, andHinf I (Promega, Madison, WI), respectively, for 4 hat 37ºC. The digested products were then elec-trophoresed on 3 per cent agarose gel containingethidium bromide (0.5 µg/ml). The serotypes werethen identified by analysing the combination of thecleavage pattern of the three enzyme digests.

Results

EpidemiologySeasonal patterns of respiratory adenoviral infec-

tions. Of 4333 children, adenoviruses were isolatedfrom 317 patients with an isolation rate of 7.67 percent. The monthly distribution of respiratory aden-oviral infection is shown in Fig. 1. Adenovirus wasdetected throughout the year; however, twooutbreaks were identified in September 2001 andDecember 2002. Adenovirus serotype analysis wasperformed by PCR-RFLP in 186 samples. Becauseof the limitation of this technique, the serotype ofadenovirus type 1, type 5, and type 6 could not bedifferentiated, and it is shown as type 1.5.6. Yearlydistribution of adenovirus serotypes in 2001–2002 isshown in Fig. 2. In 2001, adenovirus type 4 was themajor type (57 per cent), followed by type 1.5.6 (15

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280 Journal of Tropical Pediatrics Vol. 50, No. 5

FIG. 1. Monthly distribution of respiratory adenoviral infection at KMHK Hospital in 2001–2002.

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per cent), type 2 (13 per cent), type 14 (8 per cent),type 3 (5 per cent), and type 7 (2 per cent). In 2002,type 3 became the major type (46 per cent), whereasthe previous predominant type 4 decreased to 6 percent. Adenovirus type 7, which had re-emerged in1999 and declined in 2001,4 increased to 19 per centin 2002.

Demographic characteristics. For 317 patients, themean patient age was 4 years 7 months and 84.2 percent of the patients were less than 8 years old. Theage distribution of patients with different adenoviralserotype at the time of hospital admission ispresented in Table 1. For all adenoviral serotypespresented here, children between 4 and 8 years oldwere the most common group of patients with respi-ratory adenovirus infections. For serotype of aden-ovirus type 1.5.6 and type 2, the peak age wasbetween 1 and 4 years old. The ratio of male tofemale was 1.26:1 (Table 1).

Clinical manifestationsSymptoms and signs. In 317 patients, fever (body

temperature ≥ 37.5ºC) was recorded for 98.7 per centof patients. The highest body temperature was 41ºC,and the mean level of fever was 39.2ºC. The meanduration of fever was 4.8 days (range 0–19 days).According to the medical records, the othersymptoms and signs included cough (77.6 per cent),abnormal breathing sounds (crackles and/orwheezing 23.3 per cent), diarrhea (25.2 per cent),vomiting (21.8 per cent), and abdominal pain (18.9per cent). The cases of 26 patients (8.2 per cent) werecomplicated with acute otitis media. Conjunctivitiswas noted in 13.6 per cent of patients. Twenty-five(7.9 per cent) patients had skin rashes including 13maculopapules, six papules, five vesicles, and oneurticaria. Ten (3.1 per cent) patients had seizureattack, all accompanied with fever. Only one patienthad mental alteration transiently on admission andsoon recovered after supportive care. In the 186

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FIG. 2. Proportion of different serotypes of respiratory adenoviral infections at KMHK Hospital in 2001–2002.

TABLE 1Age and sex distribution of cases of respiratory adenoviral infection at KMHK Hospital in 2001–2002

Adenoviral Age group (years) Male/femaleserotype ————————————————————————————————————————— ratio

≤ 1 1–4 4–8 8–12 > 12

Type 1.5.6 8 14 6 0 0 1.5Type 2 4 14 3 0 0 0.9Type 3 1 8 23 2 0 1.1Type 4 3 15 33 24 0 1.3Type 7 1 4 7 2 0 1.3Type 14 0 3 7 3 1 1.3

Total (%) 17 (9.1) 58 (31.1) 79 (42.5) 31 (16.7) 1 (0.5) 1.26

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cases in which the serotype was analysed, pharyn-gitis and tonsillitis (47.8 per cent) were the mostcommon presentation (Table 2). Pneumonia was thesecond most common clinical diagnosis (25.2 percent). Although pharyngoconjunctival fever (PCF)was the most known adenoviral infection, there wereonly 7.6 per cent in this study. All three bronchioli-tis patients were infected with adenovirus type 2.Seven patients (3.8 per cent) had typical oral ulcera-tions of herpangina with initial impression ofenteroviral infection.

Laboratory findings. For all 317 patients, mostwhite blood cells (WBC) counts were in the normalrange. For 23.2 per cent of patients WBC countswere more than 15 000/mm3. The highest WBCcount was 33 640/mm3 with negative bacterialculture. Only five patients (1.6 per cent) hadleukopenia (WBC counts less than 4000 /mm3), withone accompanied by mild degree thrombocytopenia.No anemia and thrombocytosis were noted. C-reactive protein (CRP) levels covered a wide range:7.7 per cent of patients were in the normal range (lessthan 5 mg/l in our laboratory) and 35.8 per cent weremore than 60 mg/l. Liver enzyme, alanine amino-transferase (ALT), or aspartate aminotransferase(AST) levels were in the normal range in almost allpatients. Urinary abnormalities, such as hematuriaand/or proteinuria, were observed in 7–11.4 per centof patients. One 2-year-old boy who was infectedwith adenovirus type 4 suffered from hemorrhagiccystitis and recovered in 1 week. Cerebrospinal fluidspecimens were obtained from three children withsymptoms of central nervous system infection, andWBC counts, glucose and protein values were notelevated in any of these patients. Eleven patients hadmixed infections with other bacteria. There were sixStreptococcus, three Hemophilus influenzae, oneStaphylococcus, and one Pseudomonas aeruginosa.

Treatment and courses. Our patients all had a goodprognosis. The median days of admission was 6 days(range 2–20 days). Almost all febrile patients (91.2per cent) were treated with one or two kinds ofantibiotics (first generation of cephalosporin,erythromycin, or amoxycillin-clavulanic acid) beforethe etiological agents, the adenoviruses, wereidentified. After fever subsided, some of thesepatients suffered from persistent and prolonged(more than 2 weeks) cough that made parentsextremely anxious. One 5-year-old girl infected withadenovirus type 4 initially presented with acutepharyngitis, but disseminated intravascular coagu-lopathy developed later and resulted in petechiae,upper gastrointestinal bleeding, and tarry stool. Shewas sent to the intensive care unit for 5 days andrecovered without sequelae.

DiscussionIn the surveillance of respiratory adenovirus fromKMUH Virology Laboratory, seven serotypes (type1, 2, 3, 4, 7, 8, and 14) were observed between 1981and 2001 in southern Taiwan.4 The virus isolationrate of adenovirus increased gradually and higherthan that of enterovirus since 1999. During thecontinuous surveillance of the same programme in2001–2002, we noted there were outbreaks insouthern Kaohsiung area where the KMHK Hospitalis located. In this study, the major adenoviralserotype was type 4 in 2001 and type 3 in 2002,although adenovirus was detected throughout theyear. From the report by Tsai,7 adenoviruses are thethird viral agent (3.3 per cent) after enterovirus (10.3per cent) and respiratory syncytial virus (4.4 percent) in children hospitalized due to respiratoryinfections in Taiwan between 1997 and 1999, but theydid not report on adenovirus serotype. In the UnitedStates, adenovirus type 4 and type 7 have been the

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TABLE 2Main diagnosis and laboratory data of hospitalized children with respiratory adenoviral infection at KMHK

Hospital in 2001–2002

Main diagnosis Adenoviral serotype——————————————————————————————————————————————————

Type 1.5.6 Type 2 Type 3 Type 4 Type 7 Type 14 Total(n = 28) (n = 21) (n = 34) (n = 75) (n = 14) (n = 14) (n = 186)

Pneumonia 10 (35.7) 4 (19.0) 7 (20.6) 24 (32.0) 1 (7.1) 1 (7.1) 47 (25.2)Bronchiolitis 0 (0) 3 (14.3) 0 (0) 0 (0) 0 (0) 0 (0) 3 (1.6)Bronchitis 4 (14.3) 2 (9.5) 7 (20.5) 6 (8.0) 1 (7.1) 4 (28.6) 24 (12.9)Pharyngitis + tonsillitis 12 (42.9) 10 (47.6) 16 (47.1) 33 (44.0) 9 (64.4) 9 (64.3) 89 (47.8)PCF 0 (0) 0 (0) 4 (11.8) 9 (12.0) 1 (7.1) 0 (0) 14 (7.6)Seizure 1 (3.6) 1 (4.8) 0 (0) 0 (0) 0 (0) 0 (0) 2 (1.1)Herpangina 1 (3.6) 1 (4.8) 0 (0) 3 (4.0) 2 (14.3) 0 (0) 7 (3.8)

Percentages given in parentheses.

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significant causes of acute respiratory diseases in USmilitary trainees in 1997–1998 and 2000 outbreaks.8,9

Epidemics of adenovirus type 4 and type 7 were alsoobserved in Greater Manchester, UK.10 In the reportof lower respiratory tract infections due to adeno-virus in hospitalized Korean children, the principaladenovirus serotypes were type 7 (41 per cent), type3 (15 per cent), and type 2 (15 per cent) from 1990to 1998. The mortality rate was 19 per cent amongpatients with type 7 infection.11 On the other hand,the fatality rate for type 7 reached 28.6 per cent inArgentine children from 1991 to 1992.12 Fortunately,the clinical symptoms and signs were less severe withtype 7 infection in our study than reports of hospi-talized Korean children and Argentine children. Ourcases with adenovirus type 7 infections mostlypresented as upper respiratory infection, such aspharyngitis and tonsillitis (64.4 per cent), with a fewcases with lower respiratory infection. But pneu-monia and bronchitis occurred in 50 per cent ofpatients infected with type 1.5.6 (Table 2). There wasno mortality in this study. The severity of ourpatients was milder than that of Korean children andArgentine children at the medical center. BecauseKMHK Hospital is a community hospital, thepatients we cared for were not as severe as in themedical center. Even though one patient neededintensive care, the prognosis was better than that ofthe Korean report. In Taiwan, there was no reportdescribing the epidemiology or clinical features forrespiratory adenoviral infection. We could not,therefore, compare the difference of the incidencerate and the clinical features in respiratory adenovi-ral infection between the medical center and acommunity hospital like KMHK Hospital, indifferent areas of Taiwan.

The peak age of respiratory adenoviral infectionwas between 4 and 8 years in this study (Table 1),while the other reports showed the peak age to beyounger than 4 years old.7,11,13 The reason may be thecloser contact in kindergartens and preliminaryschools between 4- and 8-year-old children in Taiwan.In the Tsai report in Taiwan, adenoviral infectionswere more common in 1–3-year-olds in 1997–1999.7

In the study in Greater Manchester, UK(1982–1996)10 and Korea (1990–1998),11 most aden-oviral infections occurred among children less than 5years of age. In an epidemiological report of Japanin 1997, adenovirus type 7 infected cases for 0–4-year-olds accounted for 56 per cent, followed bythose aged 5–9 years (35 per cent).14 Males weremore frequently infected than females with allserotypes of adenoviruses,10–13 although our studydid not show a statistically significant difference insex distribution.

Our study showed there were 23.2 per cent ofpatients with leukocytosis without detectablebacterial infection. It is interesting that this phenom-enon seems to be specific to adenoviral infectionbecause the majority of children with RSV, para-influenza, or influenza infections have a total WBCcount less than 1.5 � 103/mm3 whereas about 25–50per cent of the children with adenoviral infectionshave a total WBC count exceeding 1.5 � 103/mm3.2

Leukocytosis associated with adenoviral infectionswere reported as early as 195415 and 1956.16

This epidemiological study may provide valuableinformation that helps physicians to obtain a clinicaldifferential diagnosis and provide treatment forrespiratory adenoviral infection in children insouthern Taiwan. The molecular surveillance ofadenoviruses should be under continuous investi-gation.

References1. Cherry JD. Adenoviruses. In: Feigin RD, Cherry JD (eds),

Textbook of Pediatric Infectious Diseases. W.B. SaundersCompany, Philadelphia, 1998; Vol. 2: 1666–84.

2. Ruuskanen O, Meurman O, Sarkkinen H. Adenoviral diseasesin children: A study of 105 cases. Pediatrics 1985; 76: 79–83.

3. Suzuki N, Ueno T, Yemashita T, Fujinaga K. Grouping of aden-oviruses and identification of restriction endonuclease cleavagepatterns of adenovirus DNA using infected cell DNA: Simpleand practical methods. Microbiol Immunol 1981; 25: 1291–301.

4. Lin YC, Ke GM, Chen HL, et al. Two decades surveillance ofrespiratory adenovirus in southern Taiwan: the reemerging ofadenovirus types 7 and 4. J Med Virol 2004; 73: 274–79.

5. Saito-Inagawa W, Oshima A, Aoki K, et al. Rapid diagnosis of

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TABLE 3Laboratory data of hospitalized children with respiratory adenoviral infection at KMHK Hospital in 2001–2002

Lab. data Adenoviral serotype(mean ± SD) ——————————————————————————————————————————————————

Type 1.5.6 Type 2 Type 3 Type 4 Type 7 Type 14

WBC count (� 103/mm3) 12.3 ± 4.7 4.5 ± 2.4 10.7 ± 4.1 11.9 ± 4.5 11.6 ± 3.2 9.0 ± 3.8Hb (g/dl) 11.5 ± 0.8 11.6 ± 0.6 11.5 ± 1.0 11.6 ± 1.0 11.1 ± 1.0 11.6 ± 0.5Platelet count (� 103/mm3) 299.0 ± 98.1 292.0 ± 77.4 236.9 ± 61.4 255.8 ± 78.4 258.8 ± 88.2 221.1 ± 52.4CRP (mg/l) 30.4 ± 35.1 24.1 ± 21.9 62.1 ± 36.8 54.2 ± 37.1 43.7 ± 34.5 45.7 ± 31.9AST (IU/l) 36.7 ± 7.4 39.2 ± 8.8 29.2 ± 6.8 29.6 ± 8.6 32.7 ± 7.0 34.2 ± 12.1ALT (IU/l) 16.5 ± 6.9 20.1 ± 9.1 12.3 ± 7.5 13.2 ± 5.6 17.8 ± 13.2 14.2 ± 5.6

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infections due to adenovirus in hospitalized Korean children:epidemiology, clinical features, and prognosis. Clin Infect Dis2001; 32: 1423–29.

12. Carballal G, Videla C, Misirlian A, Requeijo PV, Aguilar MdC.Adenovirus type 7 associated with severe and fatal acute lowerrespiratory infections in Argentine children. BMC Pediatrics2002; 2: 6–12.

13. Schmitz H, Wigand R, Heinrich W. Worldwide epidemiology ofhuman adenovirus infections. Am J Epidemiol 1983; 117:455–66.

14. Yamadera S, Yamashita K, Akatsuka M, Kato N, Inouye S.Trend of adenovirus type 7 infection, an emerging disease inJapan. Jpn J Med Sci Biol 1998; 51: 43–51.

15. Parrott RH, Rowe WP, Huebner RJ. Outbreak of febrilepharyngitis and conjunctivitis associated with type 4 adenoidal-pharyngeal-conjunctival virus infection. N Engl J Med 1954;251: 1087–90.

16. Dascomb HE, Hilleman MR. Clinical and laboratory studies inpatients with respiratory disease caused by adenoviruses (RI-APC-agents). Am J Med 1956; 21: 161–74.

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