Originalia

T. Popow-Kraupp, C. Kunz, E. Huber

Respiratory Virus Infection in Hospitalized Children 1979-1982. Diagnosis by Immunofluorescence

in Austria

Summary: Nasal secretions from 349 Austrian children under six years of age who were hospitalized for respi- ratory illnesses were screened for the presence of res- piratory syncytial virus (RSV), parainfluenza virus 1 and 3, adenovirus and influenza A virus over a period of four years by the immunofluorescence technique. 35% of the specimens were found to be positive for one of the five viruses investigated. RSV was detected in 31% of the nasal secretions and was thus the most frequently encountered causative agent of respiratory infections in the age group investigated. RSV infec- tions occurred almost exclusively in the winter months and were mainly associated with bronchiolitis and pneumonia. Only sporadic infections were found with one of the other viruses investigated.

Zusammenfassung: Diagnose yon Virusinfektionen des Respirationstraktes hospitalisierter Kinder in Osterreich yon 1979 bis 1982 mit der Immunfluoreszenztechnik. In einem Zeitraum von vier Jahren wurden mit der Im- munfluoreszenztechnik Nasensekretproben von 349 6sterreichischen Kindern unter sechs Jahren, die we- gen einer Erkrankung des Respirationstraktes hospita- lisiert werden mul3ten, auf folgende Viren untersucht: Respiratory syncytial-Virus (RSV), Parainfluenza-Vi- rus 1 und 3, Adenovirus und Influenza A-Virus. Eines dieser fiinf Viren konnte in 35% der Proben nachge- wiesen werden. RSV wurde in 31% der Nasensekrete gefunden und war daher die h~iufigste Ursache respira- torischer Infekte in dieser Altersgruppe. Infektionen mit RSV wurden fast ausschliel31ich in den Wintermo- naten beobachtet , wobei Bronchiolitis und Pneumonie die hfiufigsten klinischen Diagnosen waren. Infektio- nen mit einem der anderen untersuchten Viren wurden in diesem Zeitraum nur sehr vereinzelt nachgewiesen.

Introduction

Respiratory infections are very common in infants and toddlers and account for the majority of pediatric consul- tations and hospitalizations. Viruses have been identified in 1/3 to 1/2 of all admissions for pediatric respiratory dis- ease (1, 2). Respiratory syncytial virus (RSV) has repeat- edly been shown to be the most important pathogen in children up to five years of age. It causes 60-90% of bron- chiolitis cases and 5-40% of pneumonia cases (3-6). Oth- er important respiratory pathogens in this age group are parainfluenza virus type 1, the most common cause of acute laryngitis (croup), parainfluenza virus type 3, adeno-

viruses and influenza virus type A ( t , 4, 6-13). Further- more, there is some evidence that lower respiratory tract infections may predispose to recurrent attacks of wheez- ing and chronic pulmonary changes later in life (14, 15). In recent years the diagnosis of viral respiratory disease has been facilitated by the introduction of the immuno- fluorescence technique (IFT) (16). This method identifies the viral pathogen in cells of nasopharyngeal secretions within hours, thus providing a rational means for the management and control of infection. The excellent correlation between the IFF and virus isolation in cell cul- ture has been shown in several independent studies (1%19). In addition to its rapidity and specificity, the IFT is far less laborious and time-consuming than virus iso- lation and has therefore become the method of choice in studying the epidemiologic behaviour of respiratory virus- es (8, 20, 21). We introduced the IF/" in our laboratory in 1978. The aim of this prospective study was to determine the incidence, the temporal distribution and some clinical aspects of res- piratory viral infections in hospitalized Austrian infants and young children.

Patients and Methods

Patients

From January 1979 until December 1982, nasopharyngeal aspi- rates were obtained from 349 children (206 males, 143 females) under six years of age who were admitted with any form of respi- yatory illness. A respiratory tract infection was the reason for hospitalization in 319 patients. Twenty-six children were hospi- ,talized for other reasons, but had respiratory symptoms on the day of admission. Four patients were newborns and acquired pneumonia during their stay in the nursery. Specimens were ob- tained from patients in the Departments of Pediatrics of the Universities of Vienna, Graz and Innsbruck, and from Chil- dren's Hospitals in Vienna and Salzburg. Viennese hospitals serve predominantly an urban population and the pediatric hos- pitals in the provincial capitals an urban population and the sur- rounding rural areas. A uniform questionnaire was sent to all participating hospitals and had to be filled in by the clinician for each individual pa- tient. This questionnaire was sent to our laboratory with the specimen and gave the following information: name, age, sex, date of the onset of symptoms and of hospitalization and clinical symptoms. Each illness had to be assigned to one of the four clinical categories: rhinopharyngitis, laryngotracheobronchitis, bronchiolitis and pneumonia. The clinicians were invited to give further information on individual clinical details.

Received: 29 September 1983/Accepted: 5 January 1984

Dr. Therese Popow-Kraupp, Prof. Dr. C. Kunz, Institute of Virology, University of Vienna, Kinderspitalgasse I5, A-1095 Vienna; Prof'. Dr. E. Huber, Landeskrankenanstalten Salzburg, Kinderspital, Milliner Hauptstrage 48, A-5020 Salzburg.

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T. Popow-Kraupp et al.: Respiratory Virus Infections in Austria

Methods'

Specimens were screened for the presence of the following viral antigens: RSV, influenza virus A and parainfluenza viruses 1 and 3. Adenoviruses were included in the screening program in November 198]. The indirect immunofluorescence method according to Gardner and McQuillin (t6) was used for the detection of viral antigens in cells of nasopharyngeal secretions.

Specimen Collection and processing: Nasopharyngeal aspirates were obtained by using a tracheal suction kit (Nunc, Kamstrup, Denmark). A nasal catheter was attached to one of the two out- lets of the suction set, while the other was connected to a suction pump. With the suction turned on, the nasal catheter was passed through each nostril and nasopharyngeal secretions were sucked into the tube. An adequate quantity of secretion (minimum 0.2-0.3 ml) was usually obtained within a few seconds. Tubes containing the secretions were immediately transported in melt- ing ice to the laboratory. The nasopharyngeal secretion from each patient was dispersed in 3 ml of phosphate-buffered saline (PBS), pH 7.2, by gently pipetting with a wide bore Pasteur pi- pette. Thick fragments of mucus that would not break up were discarded. The cells were separated by centrifugation at 350 g for 10 min at 4 ° C. The supernatant was removed and the cellu- lar pellet washed with PBS and resuspended in a sufficient amount of PBS (usually 0.5-1 ml) to dilute any mucus still pre- sent and to make a sufficient number of preparations with a sat- isfactory amount of cells on the slides to test for the different vi- ral antigens. 20 ~1 of the cell suspension were spread evenly in each of the areas of polytetrafluoroethylene-coated multispot microscope slides (C. A. Hendley & Co., Essex, England). Nasopharyngeal secretion smears were air-dried and fixed in acetone for 10 min. Slides were held at 4 ° C until the staining procedure was started. Specimens from pediatric hospitals in the provincial capitals were prepared there and the acetone-fixed slide preparations of the cells were sent to our laboratory.

Immune reagents: Calf immune sera against RSV and Influenza A virus were obtained from Wellcome Reagents Ltd., rabbit im- mune serum against parainfluenza virus 1 and adenovirus (group-specific) was kindly provided by Prof. M. Grandien (Sta- tens Bakteriologiska Laboratorium, Stockholm) and parain- fluenza virus 3 antiserum by Prof. P. S. Gardner (Dept. of Vi- rology, Newcastle upon Tyne). The fluorescein isothiocyanate (FITC) conjugated anti-bovine and anti-rabbit immunoglobulin from Wellcome Reagents Ltd. was Used. All reagents were standardized and quality checked by the Cen- tral Public Health Laboratory, Collindale, according to the in- structions of Gardner and McQuillin (16). Immunofluorescence staining procedure and microscopy: The different areas of a slide preparation from one nasopharyngeal secretion were each overlaid with one of the five viral antisera, incubated in a moist chamber at 37 ° C for 1 h and then rinsed for 30 min in three changes of PBS. FITC conjugated anti-rabbit and anti-bovine immunoglobutin diluted 1:10 in PBS with 0.005% Evansbtue counterstain was then added and the slides were incubated for 30 min at 37 ° C. This was followed by a 30 min rinse in three changes of PBS and a final wash in distilled water for 2 rain. The slides were mounted with Glycerol-PBS (9:1) and examined with a Leitz SM-Lux fluorescence micro- scope, fitted with an HBO 50 W mercury vapor lamp, a BP 450-490 exciter filter and an LP 515 barrier filter. Acetone-fixed slide preparations of virus infected and uninfected tissue culture cells were used as controls.

A nasopharyngeal secretion was considered positive for one of the five viruses investigated if 1) at least three cells showed the characteristic fluorescence specific for the corresponding virus and 2) the results for the other antigens tested were negative. In the first year of the study, fixed and unstained slide prepara- tions from 41 patients were sent to the Department of Virology in Newcastle upon Tyne, the reference laboratory for the diag- nosis of viral respiratory diseases by the IFF of the European Group for Rapid Viral Diagnosis, for confirmation of results. The agreement between the test results of both laboratories was 100%: 25 specimens were negative for all viruses investigated, 15 nasopharyngeal secretions were positive for RSV and one for parainfluenza virus 3. Statistical analysis of the sex distribution of RSV infected and uninfected children was performed using the chi square test.

Results

The t e m p o r a l d i s t r ibu t ion of the spec imens inves t iga ted and the test results a re shown in F igure 1. The m a j o r i t y of the spec imens (61%) were sent to o u r l abo ra to ry in the

1979 4 0

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2 0

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1 2 3 4 5 6 7 8 910 1112 m o n t h s

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"6 d z

4 0

3 0

2 0

10

4 0

3 0

2 0

1 0

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

1 9 8 0

months

1981

1 2 3 4 5 6 7 8 91011 12 months

4 0

3 0

2 0

1982

1 0

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 months

Figure 1" Temporal distr ibution of respiratory syncyt ia l vi- rus (B), parainfluenza virus 1 and 3 ([]), adenovirus ([]) and influenza virus A (E3) positive specimens from 1979 to 1982.

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winter months (December-March). 122 specimens (35%) were positive for one of the five viruses investigated. RSV was detected in 1(19 nasal secretions (31%) and was thus the most frequently encountered virus in the study. Dis- tinct outbreaks of RSV infections occurred in the winters of 1979 and 1980/81 with peak incidences in February 1979 (75% RSV positive), February 1981 (61% RSV posi- tive) and in the early spring of 1982, when the outbreak peaked in April (75% RSV positive). Only sporadic cases of RSV infections occurred in the winter of 1979/8(I. In August 1980, pneumonia developed in 20 newborns in the nursery of one of the gynecological departments. Of the four specimens sent to our laboratory, RSV was detected in three, while one was RSV negative; this specimen was not collected until Day t4 after the onset of symptoms, however. With the exception of this summer outbreak, the virus was only found during the colder months of the year. Only sporadic cases of infections with parainfluenza and adenoviruses and only one case of influenza A virus infec- tion were detected in the entire study period. No epide- miological differences were found between the provincial capitals and Vienna.

Table 1 : The number of specimens and positive test results in the different age groups.

0--6 mo 129 (38) 62 (48) 3 (2) 65 (50) 6 mo-I yr 59 (17) 18 (31) 6 (10) 24 (41) 1-2 yr 72 (21) 20 (28) 2 (3) 22 (31) 2-3 yr 30 (9) 2 (7) ~ (3) 3 (10) .3(,yr 53 (15) 7 (13) 1 (2) 8 (15)

AD = adenovirus; P1 = parainfluenza virus 1 P3 := parainfluenza virus 3, IA = influenza A virus.

Table 1 summarizes the results by age groups. The major- ity of specimens was obtained from the age group 0 ~ months, and the highest percentage of RSV positive test results was also found in this group. The frequency of RSV infections decreased with increasing age. The only patient with an influenza A virus infection was a 5-year- old girl. The relation between the age distribution and the clinical diagnosis is shown in Figure 2. Detailed information on age, sex and clinical diagnosis was available in 343 (204 males, 139 females) of the 349 patients. The main reasons for hospitalization of children with upper respiratory tract symptoms were: high lever (62%), febrile convulsions (7%), otitis media (14%), congenital abnormalities (14%) and meningitis (3%). A virus was found in 18% of the patients with upper respiratory tract symptoms. In children aged 0-6 months, bronchiotitis was the most prominent clinical diagnosis, and in 64% RSV was the causative agent. The viral etiology was proven in 56% of the cases with pneumonia and in 47~ of the cases with la- ryngotracheobronchitis in this age group. Bronchiolitis

Z

9

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o

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Figure 2: The number of respiratory syncytial virus ( I ) , pa- rainfluenza virus 1 and 3 (lltl), adenovJrus ([]) and influenza virus A (~t) positive specimens in relation to age and clin- ical diagnosis.

was seen less frequently in older children, but the per- centage of RSV positive specimens remained high up to two years of age.The frequency of RSV infections in chil- dren with laryngotracheobronchitis and pneumonia de- creased with increasing age. RSV infections did not seem to be associated with febrile convulsions, since none of the specimens from these eight patients revealed the presence of RSV antigen, and only one of the eight was positive for parainfluenza virus 3. On the other hand, RSV was frequently associated with otitis media, i.e. five of the 15 nasopharyngeal secretions (33%) from patients with respiratory tract infections com- plicated by otitis media were positive for RSV. Of the 343 children admitted with respiratory infections, 204 were boys and 139 girls. Nearly the same proportion of specimens from each sex were positive for RSV, i.e. 34% of those from males and 29% of those from females. The same proportkm of each sex (90%) had RSV infec- tions of the lower respiratory tract. We also analyzed the number of virus-positive specimens in relation to the number of days after the onset of symp- toms (Figure 3). Information concerning the onset of ill- ness was available for 294 patients. 62% of the virus-posi-

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T. Popow-Kraupp et al.: Respiratory Virus Infections in Austria

.9 4o- 3 0 -

~ 20-

~ tO )

c 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

n of Clays after the onset of symptoms

tive specimens were drawn within the first week after the onset of symptoms. In some cases, RSV could be detected up to the third week of illness.

Discussion

The immunofluorescence technique is very sensitive when highly specific and quality controlled antisera are em- ployed (1~19). The diagnosis of viral disease may be established six hours after the specimen arrives in the laboratory, thus enabling a more accurate management of the patient. Furthermore, the IFT is especially suitable for epidemiological studies, particularly in infants and young children. A more accurate measure of the total number of viral infections is probably obtained by the IFI" than by virus isolation or serological techniques. Respira- tory viruses are very labile; therefore, reliable figures on the prevalence of respiratory viruses may only be obtain- ed by tissue culture isolation procedures when specimens arc inoculated immediately after sampling or when they are transt~rted quickly in melting ice to the virus labora- tory. Slides prepared for staining by the fluorescent anti- body technique, however, can be transported over long distances without influencing the test results (22). Sero- logical methods such as complement fixation tests and tis- sue culture neutralization techniques are relatively ineffi- cient for the detection of RSV and parainfluenza virus 1 infections in infants and young children (21, 23, 24). The IFT does have some disadvantages, however: prepar- ing the slides is laborious and requires skill. Therefore, hospitals and practitioners in areas at a certain distance from a virus laboratory are at a disadvantage if they are not able to prepare the slides themselves. Furthermore, microscopic screening is time-consuming, especially when large numbers of specimens are to be examined. Some of the infections may be missed due to inadequate sampling which results in an insufficient amount of nasal secretion. However, the many advantages of the IFT outweigh these shortcomings. In our study, one of the five viruses investigated was de- tected in approximately ~/3 of the children hospitalized with an infection of the respiratory tract. RSV was by far the most frequently encountered virus and was the major cause of bronchiolitis and pneumonia, as has been shown repeatedly by various groups in other countries (3-6). The predominant appearance of RSV in the colder months of the year is in agreement with several other studies on the epidemiological behaviour of this virus (6, 8, 21, 25).

18 19

n = 2 9 4

r-!

20 21 =,21

Figure 3: The number of respiratory syncytial virus (11), parainfluenza virus 1 and 3, adenovirus and influen- za virus A (E;) positive specimens in relation to the number of days after the onset of symptoms.

The RSV outbreak in the nursery of one of the gynecolog- ical departments in August 1980 might have been due to the practice of partial rooming-in, whereby the infants spend several hours with the mother and the rest of the day in the nursery. The virus was apparently brought in by a visitor, staff member or by one of the mothers, and the infection was spread from one child to the next. This demonstrates the high pathogenicity of RSV for newborn infants and the fact that nosocomial infections with this vi- rus can pose considerable problems on pediatric wards (26-28). Rapid identification of RSV and other respirato- ry viral infections in patients and staff by the IFT can aid in reducing the risks of cross-infection by rapidly separat- ing the virus excretors from the other infants, particularly from those with congenital abnormalities and immuno- suppressive therapy. Only sporadic infections were found with one of the other viruses investigated; thus, conclusions on the epidemio- logical behaviour and clinical details of these viruses can- not be drawn. In contrast to studies from other countries (2, 4, 8, 9), only very few cases of parainfluenza virus I in- fections were detected. This might reflect the local epide- miological pattern of this virus in the past tour years. The low incidence of parainfluenza virus 1 infection may also be explained by the fact that children with acute laryngitis are usually treated in the ENT departments where sam- ples for virological studies were not taken. In the study period, only a single case of influenza A virus infection was found (December 1982). This might be ex- plained by the fact that the incidence of influenza A virus infection in the general population was very low during the study period (data from the influenza surveillance sys- tem of the city of Vienna). No large outbreaks and only sporadic cases occurred in Austria in these four years. Furthermore. 74% of our patients were under the age of two years when the clinical picture of influenza A virus in- fection tends to be mild and usually does not require hos- pitalization (29). In our study, the age distribution of children with RSV in- fection was similar to that found in other studies (3-5, 25). Of children admitted with RSV infection, 56% were less than six months old, an age when most infants possess maternal antibodies against this virus. In this group, RSV was particularly associated with bronchiolitis and pneu- monia. Severe infection with RSV requiring hospital- ization occurs predominantly in primary infection which is usually acquired before the age of two years. Reinfections with this virus occur frequently, but the illnesses caused are usually milder. This seems to confirm the assumption

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that acquired local neutralizing IgA antibodies play a ma- jor role in the defense mechanisms against RSV infection (30, 31). The best chance to obtain a virus-positive result is given when samples are drawn within the first week after the onset of symptoms. Nevertheless, prolonged virus excre- tion up to the third week has been described for young children with lower respiratory tract disease (32). Our three RSV positive patients, whose specimens were drawn on Days 20 and 21 of their illness, belong to this group of patients. In summary, we have described the incidence, temporal

distribution and some of the clinical aspects of RSV infec- tions over a four-year period in Austrian children hospital- ized for respiratory disease. The IFT, also in our hands, has been shown to be a very useful tool which helped to detect the causative agent in 35% of the infections of the respiratory tract.

AcRnowledgements We wish to thank Mrs. J. McQuillin for the comparative analysis of some of our samples. We also wish to thank Mrs. L Hartnett for her ex- Cellent technical assistance and Mag. U. Apfelthaler for typing the ma- nuscript. This study was funded by the "Medizinisch-wissenschafllicher Fonds des Btirgermeisters der Bundeshauptstadt Wien ' .

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