Viral Lower Respiratory Tract Infections in Filipino Children Petri Ruutu, Pekka Halonen, OUi Meurrnan, Clotilde Torres, Fern Paladin, Kunio Yamaoka, and Thelma E. Tupasi
From the Second Department of Medicine, Helsinki University Central Hospital, and the Department of Virology, University of Turku, Finland, and the Research Institute for Tropical Medicine, Department of Health, Manila, Philippines
It has been estimated that ~4 ,000,000 children in developing countries die each year from pneumonia . Most of these pneumonias are believed to be caused by bacteria since a number of studies using lung puncture aspirates have demonstrated growth of bacteria in more than half of the lung aspirates examined . In developed countries, a large proportion of pneumonias in children are caused by viruses. Some viruses are known to affect the defensive mechanisms of the lung against bacterial infection [3, 4], thus possibly predisposing to bacterial superinfections. In developing countries, viruses other than measles have been associated with 14%-47% of pneumonia cases in children [2, 5]. Studies using lung puncture aspirates and viral diagnostics have revealed viral infection in 0-50% of children with pneumonia [6-8], and recently Shann et a1.  isolated a virus in 18 (29%) of 62 lung aspirates from children with severe pneumonia in Papua New Guinea. Antigens of respiratory viruses can now be detected in respiratory secretions by immunofluorescence  and enzyme immunoassay (EIA) . Solid-phase EIA are particularly practical when specimens are collected in developing countries. Viral antigens are relatively stable in respiratory secretions, and the specimens can be stored a day or two without cooling. In addition, EIA methods have proved superior in the serologic diagnosis of virus infections compared with the complement fixation methods used previously [12, 13]. In the present study, virus isolation, EIA antigen detection,
Received 9 December 1988; revised 24 August 1989. Supported by a grant from the Academy of Finland. Reprints and correspondence: Dr. Petri Rnutu, Second Department ofMedicine, Helsinki University Central Hospital, SF-290 Helsinki, Finland. The Journal of Infectious Diseases 1990;161:175-179 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6102-0002$01.00
and EIA serology were used to determine the viral causes of acute lower respiratory tract infection among Filipino children.
Materials and Methods Patients. All patients 4O/min (9 %) or sputum production or both. Sputum production was regarded positive if the escort or the enrolling physician described it as loose or slimy. Thirty-two patients died. The nutritional status of the patients was classified by comparing the weight of the patient with the recommended normal weights by age among Filipino children [14, 15] as severe «60% of normal), moderate (60%-75 % of normal), or mild (76%-90% of normal) malnutrition and normal (>90% of normal). Serum specimens. Blood for acute- and convalescent-phase serum specimens was collected by venipuncture from 220 patients (69 %). Half of the convalescent -phase specimens were collected by a follow-up activity mainly in periurban slums. Because of problems in tracing the patients in a highly mobile population, the interval between the acute- and convalescent-phase specimens varied: 10-15 days in 42 % of the patients, 16-30 days in J7%, 31-45 days in 14%, and 45-80 days in 7%. Nasotracheal aspirates. The catheter of a disposable mucus extractor (Vygon, Ecouen, France) was inserted through the nostril; suction was applied only after the catheter elicited a vigorous cough reflex. A sample volume of 0.5-3 ml was collected into a sterile mu-
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Viral causes of acute lower respiratory tract infection were studied prospectively between 15 June and 31 October 1984 in 312 Filipino children < 5 years old living in periurban slums and middle-class housing. The cause was based on viral antigen detection, virus isolation, and antibody assays. There were 131 children (41.2%) who were admitted to the hospital, and 150 (47%) had an infiltrate on chest radiograph. A total of 198 viral infections were confirmed in 162 patients (51.9%),42.3% with single viral infection and 9.6% with mixed (two or more) infection. The infections were measles (21.4%), influenza A (15.9%), parainfluenza types 1, 2, and 3 (8.8%), respiratory syncytial virus (7.1%), influenza B (5.8%), enteroviruses (5.1%), adenoviruses (39%), herpes simplex virus (1.6%), and cytomegalovirus (1.3%). Viral infections other than measles were seen in 39.7% ofthe cases. The presence of viral infection correlated with better nutritional status. Influenza A or B diagnosis was associated with mild forms of acute respiratory tract infection, measles and a preceding rash with severe disease.
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Ruutu et al.
cough and/or sore throat and/or nasal secretions
~ breathing frequency ~40/mln and/or sputum production and/or wheezing and/or rales and/or percussion dullness and/or Infiltrate on the chest x-ray
NO .----~ AURI
~YES ches~aln and/or chest In rawlng and/or cyanosis
Figure 1. Decisions used to enroll patients with acute lower respiratory tract infection (ALRI). AURI = acute upper respiratory tract infection.
cus trap, and at least 0.2 ml of the secretion was immediately mixed with 2 ml of virus transport medium and kept refrigerated at 4°C until processed for virus isolation. The remaining aspirate was frozen at -20°C for 3-7 months and transported to Finland by air cargo on dry ice for the assay of virus antigens. Virus antibody assays. Antibodies were determined in acute- and convalescent-phase serum specimens by EIA using previously published methods against the following viruses: influenza (A and B), parainfluenza (types 1, 2, and 3), respiratory syncytial (RSV) , adenovirus, measles, cytomegalovirus, herpes simplex (HSV), and mumps [12, 13, 16, 17]. A fourfold or greater rise in the IgG antibody titer was considered diagnostic. In patients with a rise in antibody to more than one virus, IgM antibody for Epstein-Barr virus was assayed. Virus antigen detection. Viral antigens of influenza (A and B), parainfluenza (1,2, and 3), RSV, and adenoviruses (group-reacting hexon antigen) were assayed in 293 (92% of patients) nasotracheal aspirates by EIA . Virus isolation. Virus isolation was carried out in Z73 patients (86%). Nasotracheal aspirate suspended in virus transport medium was centrifuged to deposit cellular debris, and the supernatant was inoculated on confluent primary monkey kidney, HEP-2, and human embryonic lung cell monolayers grown on cell culture tubes. Unused supernatants were stored at -70°C for future use. The cultures were observed for 2 weeks for any cytopathic effect or hemadsorption reaction. Isolates were identified by immunofluorescence and other conventional methods where appropriate. Statistical analysis. The x2 test with Yates's correction or Fisher's exact test, where appropriate, was used for testing differences in rates. P ~ .05 was considered significant.
Results There were 198 viral infections diagnosed in 162 (51.9%) patients from a total of 312 patients tested (table 1). Influenza
Table 1. Viral infections in 312 Filipino children with acute lower respiratory tract infection.
Virus Influenza A Influenza B RSV Parainfluenza 1 Parainfluenza 2 Parainfluenza 3 Adenovirus Cytomegalovirus Herpes simplex Polio 1 Polio 3 Coxsackie A9 Coxsackie B5 Other enterovirus Measles
No. tested 308 308 308 308 308 308 308 308 308 273 273 273 273 273 220
* Virus isolation or antigen detection. t Calculated from those with paired sera.
No. (%) of patients with infection 49 (15.9) 18 (5.8) 22 (7.1) 13 (4.2) 3 (1.0) 11 (3.6) 12 (3.9) 4 (1.3) 5 (1.6) 5 (1.8) 1 (0.4) 2 (0.7) 2 (0.7) 4 (1.5) 47 (21.4)t 198
Virus demonstrated in nasotracheal aspirate* 28 14 11 6 1 5 3 0 0 5 1 2 2 4 2 84
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A was the most common virus, infecting 49 patients (15.9%). Measles antibody response was seen in 47 (21.4%) of220 patients with paired sera. An enterovirus was isolated in the nasotracheal aspirates of 14 patients (5.1%). Other than measles (alone or in mixed infection), 151 viral infections were seen in 124 (39.7%) of the 312 patients for whom samples for viral diagnostics were submitted. Patients with recent vaccination for measles or poliomyelitis were excluded from calculations of these viral infections. Mixed viral infections were seen in 30 patients (9.6%). Dual infections were detected in 25 patients. Evidence of three and four infections were found in 3 and 2 patients, respectively. There were 65 total infections in these 30 patients. All viruses detected in the study population were involved in mixed infections. Concomitant infection with influenza A was demonstrated in 15 (23%), measles in 14 (21.5%), parainfluenza in 14 (21.5%), and RSV in 6 (9.2 %) patients. The diagnosis was based on virus isolation or antigen detection in 12 infections. The time interval between acute- and convalescent-phase serum was not significantly longer for those with mixed infection than for those with single infection. No Epstein-Barr virus antibodies of IgM class were seen in patients with mixed infections. There were 11 patients with bacterial infection verified by blood culture (Streptococcus pneumoniae, 5; Haemophilus infiuenzae, 3; Staphylococcus aureus, 2; Salmonella typhi, 1). Of these 11, 4 had evidence of concomitant viral infection. The results of investigations for cause of bacterial infection will be reported separately in more detail. The distribution of various specific viral infections according to age did not show significant differences between age groups (data not shown). When infections verified by virus
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Respiratory Viruses in Filipino Children
RESPIRATORY SYNCmAL VIRUS
the prevalence of viral infections seemedto decline from June and July (57% and 64% of patients) to September and October (41.5% and 39.7%). The different viral infectionswereanalyzedfor featuresassociatedwith the severity of the acute lower respiratory tract infection (table 2). InfluenzaA infections were significantly lesscommon amongthoseadmitted to thehospital thanamong outpatients (P < .01, Yates's corrected X2) . Influenza B was significantly lesscommonin thosewitha respiratory frequency >40/min (P < .05, Yates's corrected X2 ) . The differences remained significant for the patients whosediagnoses weremade by virus isolation or antigen detection. Measles was seen significantly more commonly among hospitalized patients (P < .01, Yates's corrected X2) . The presenceof chest indrawing on physical examination did not correlate with any documented viral infection. In the 27 fatal cases with viral studies, five viral infectionsweredetectedby isolationor antigen detection: parainfluenza in 2 patients, RSV in 1, and enterovirus in 2. Convalescent-phase sera were not available for the fatal cases. No viral isolationswerecarried out in autopsies performed. A historyof skin rash during the previous3 weekswasalso analyzed with the clinical features associated with severity of infections, and it was statistically significantly associated with all features indicatingseveredisease(table2). One-third of the fatal cases were associated with a rash. Viral infection was diagnosed in 81 (54%) of 150 patients with an infiltrate (table 2). Virus isolation or antigen detection was positive in 26.8% of the patients with an infiltrate (data not shown). Among those with an infiltrateon chest radiograph and paired serum specimens, rise in level of measles antibody was seen in 25%. Rash was seen in 22.4 % of cases withan infiltrate. Chest radiography wasnot performed in about one-third of the cases with less severepresentation, so a comparison for this clinical feature is not possible.
20 ALL VIRUSES· VI
Month of 1984
No. patients enrolled
Figure i. Monthly distribution of viral infection in 312 patients. *Those shown plus adenovirus, cytomegalovirus, herpes simplex, and enteroviruses. tLast 2 weeks of June.
Discussion Our prospective study demonstrated a viral cause in 162 (51.9%)of 312 Filipinochildren75% of the recommended Philippinenormal weight foragethan in thosebelowthisweightlimit (41/154 vs. 23/145, P = .034, Yates's corrected x2) . Similarly, infections caused by any virus proven by virus isolation or antigen detection were significantly more common in the patients with weight >75% of the recommended normalweight (50/154 vs. 301145, P = .031, Yates's corrected X2) . Numbers of diagnoses made by all methods including serology did not differbetween patients with different nutritional status (data not shown). In contrast, rash was clearly more common among those severely and moderately malnourished than those in better nutritional status (35/153 vs. 181159, P = .008, Yates's corrected X2 ) . During the period of June to August 1984, >20% of patientsenrolled had influenza A infection(figure2), with a decline toward October. RSV demonstrated a similar trend. Measles peaked at 44 % of those tested in June. Altogether,
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Table 2. Severity of disease according to viral diagnosis. % with virus infection* Admitted to hospital