Helsinki University Biomedical Dissertations No.

7
Hospital for Children and Adolescents,
Helsinki University Central Hospital,
Helsinki, Finland

Etiology and Antimicrobial Treatment of

Pneumonia and Other Common Childhood
Infections Warranting Hospitalization
Elina Vuori-Holopainen

Academic Dissertation

To be publicly discussed with the permission of the Medical Faculty of the

University of Helsinki, in the Niilo Hallman Auditorium of the Hospital for
Children and Adolescents,
on September 28th 2001, at 12 noon.

Helsinki 2001

2
Supervised by
Professor Heikki Peltola, MD
Hospital for Children and Adolescents
Helsinki University Central Hospital
Helsinki, Finland
Reviewed by
Professor Jussi Mertsola, MD
Department of Pediatrics
Turku University Hospital
Turku, Finland
and
Professor Matti Korppi, MD
Department of Pediatrics
University of Kuopio
Kuopio, Finland
Dissertation opponent
Docent Per Ashorn, MD
Medical School
University of Tampere
Tampere, Finland

ISSN
1457-8433
ISBN (nid.) 952-10-0117-8
ISBN (pdf) 952-10-0118-6
http://ethesis.helsinki.fi
Layout: Callide/Terttu Halme
Yliopistopaino, Helsinki 2001

Kuka tietvi mist me tulemme

Ja miss on matkamme mr?
Hyv, ett me sitkin tutkimme.
Ei tutkimus ole vr.
Mut yhden me tiedmme varmaan vaan:
Me olemme kerran nyt pll maan
ja tll meidn on elminen
miten taidamme parhaiten.
- Eino Leino -

To Juha and Ronja

CONTENTS
1 ABSTRACT

2 ABBREVIATIONS

11

3 LIST OF ORIGINAL PUBLICATIONS

12

4 INTRODUCTION

13

5 REVIEW OF THE LITERATURE

5.1. Definition of lower respiratory infections
5.2. Epidemiology and pathogenesis of lower respiratory infections
5.2.1. Epidemiology
5.2.2. Pathogenesis
5.3. Pathogens associated with lower respiratory infections
5.3.1. Streptococcus pneumoniae
5.3.1.1. Serotypes and virulence factors
5.3.1.2. Carriage
5.3.1.3. Pneumococcal infections
5.3.1.4. Prevention of pneumococcal infection
5.3.2. Haemophilus influenzae
5.3.3. Moraxella catarrhalis
5.3.4. Mycoplasma pneumoniae
5.3.5. Chlamydia pneumoniae
5.3.6. Respiratory viruses
5.3.7. Mixed infections
5.4. Methods for identification of etiology
5.4.1. Bacterial agents
5.4.1.1. Culture
5.4.1.2. Antigen detection
5.4.1.3. Antibody and immune complex assays
5.4.1.3.1. Pneumococcal serology
5.4.1.3.2. Other bacterial serology
5.4.1.4. Nucleic acid detection
5.4.2. Viral agents
5.4.3. Lung tap in childhood pneumonia
5.4.3.1. Earlier clinical experience
5.4.3.2. Risks of the procedure

14
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15
16
20
20
21
21
22
22
23
23
24
24
25
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27
27
27
28
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29
30
31
32
32
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6
5.5. Acute host response parameters distinguishing viral from
bacterial etiology
5.6. Antimicrobial therapy
5.6.1. Treatment in relation to antimicrobial resistance
5.6.1.1. Streptococcus pneumoniae
5.6.1.2. Influence of -lactamase production
5.6.2. Antimicrobial treatment studies
5.6.3. Length of antimicrobial treatment
5.6.4. Current guidelines for treatment of pneumonia

33
34
35
35
36
36
37
38

6 AIMS OF THE STUDY

6.1. General aim
6.2. Specific aims

38
38
38

7 PATIENTS AND METHODS

7.1. SE-TU study (I-III)
7.1.1. Study design
7.2. KE-TU study (IV, V)
7.2.1. Study design
7.2.2. Technique of lung tap
7.3. Specimens and microbiologic methods
7.3.1. Conventional microbiology (I-V)
7.3.2. Serology (I-III)
7.3.3. Lung aspirates (IV, V)
7.3.4. Serotyping and susceptibility testing of Streptococcus
pneumoniae (V)
7.4. Criteria for etiology (I-V)
7.5. Radiologic assessment (I-V)
7.6. Statistical methods (II, III, V)

39
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42
42
42
42
44
44

8 RESULTS
8.1. Etiology in the four clinical manifestations (I)
8.2. Patients with randomized treatment (II, III)
8.2.1. Characteristics and bacterial etiology (II, III)
8.2.2. Antimicrobial therapy, length of treatment,
and recovery (II, III)
8.2.3. Clinical outcome (II, III)

47
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52

45
46
46

54

7
8.3. Lung tap (IV, V)
8.3.1. Sample validity and overall yield
8.3.2. Etiology of consolidated pneumonia
8.3.2.1. Moraxella osloensis (IV)
8.3.3. Adverse events
8.4. Diagnostics of pneumococcal pneumonia with
different methods (I, V)
8.5. Pneumococcal serotypes and antimicrobial susceptibility (V)

55
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56
57
61
62

9 DISCUSSION
9.1. Etiology of pneumonia and other suspected invasive
infections warranting hospitalization (I)
9.1.1. Study design
9.1.2. Microbiologic methods
9.1.3. Etiologic findings in relation to previous studies
9.2. Effectiveness of antimicrobial treatment (II)
9.3. Shortening of therapy (III)
9.4. Lung tap in the diagnosis of pediatric pneumonia (IV, V)
9.4.1. Representativeness of the sample
9.4.2. Positivity rate of lung tap
9.4.3. Ethical considerations and applicability of lung tap
9.5. Invasive isolates of Streptococcus pneumoniae (V)
9.6. General discussion and future considerations

63
63

10 SUMMARY AND CONCLUSIONS

74

11 ACKNOWLEDGMENTS

75

12 REFERENCES

77

ORIGINAL PAPERS

89

63
64
65
66
68
69
69
69
71
72
72

1 ABSTRACT
Background: An antimicrobial for suspected bacterial pneumonia and other common
childhood infections is generally chosen without knowledge of the causative pathogen.
The etiologic diagnosis of pneumonia is difficult, because uncontaminated samples
are not easily obtained and indirect, rapid, and accurate laboratory tests for most
pathogens are not available. More sensitive and specific diagnostic methods are
needed. The aim of the present study was to investigate the etiologic diagnostics and
treatment of common invasive bacterial infections of children, with special emphasis
on alveolar pneumonia.
Patients and methods: The effectiveness of two parenteral antimicrobial
regimens (procaine penicillin i.m. vs. cefuroxime i.v.) and two different lengths
(4 days vs. 7 days) of treatment were compared in a prospective, randomized
study comprising 154 3-month-old children with symptoms and signs suggestive of
acute invasive bacterial infection. The patients were enrolled during two time periods
from February 1988 to October 1989, and from March 1991 to October 1993.
The etiology was studied with oropharyngeal and blood cultures, viral antigen detection
tests, and serology including antibody assays of 15 viruses, seven bacteria, and one
protozoan.
Microbiologic diagnosis of childhood pneumonia was further investigated
by performing a lung tap on 34 children with consolidated community-acquired
pneumonia during a three-year study period from December 1997 to December
2000. In addition to conventional cultures, nucleic acid detection was performed
on the lung aspirates for the common respiratory pathogens. The yield obtained
by lung tap was compared with that obtained by routine noninvasive microbiologic
methods. Antimicrobial susceptibility and serotypes of Streptococcus pneumoniae
isolates were studied.
Results: The results of this study confirm the importance of S. pneumoniae as
a causative bacterial agent in common childhood infections. Serologic evidence
for a pneumococcal etiology was found in 46 % of patients with pneumonia, in
30 % of those with other respiratory infection, in 24 % of patients with sepsislike infection, and in 33% of those with other acute infection. In the treatment
of these infections, no significant difference was observed in the clinical efficacy
of parenteral penicillin as compared with cefuroxime. Recovery was similar
regardless of the regimen used, there being one potential failure in each treatment
group, and no difference in the frequency of needing a physician within 1 month
after discharge. The outcome of patients treated with 7-day course of -lactam
antimicrobials was not better than that of patients treated for 4 days. There was
one clinical failure potentially related to the shorter course of treatment.
The important role of S. pneumoniae as a cause of pneumonia was further
confirmed by studies of lung aspirates, in which it was found in 53 % (18/34) of

10
patients. In all, lung tap provided the etiologic diagnosis in 59% (20/34) of
cases. Two patients had dual bacterial infection, one had mixed bacterial-viral
infection and one had single viral infection. The procedure did not cause any
adverse events requiring treatment.
Conclusions: The results of this study suggest that in lower respiratory infections
and in many other bacterial infections in children, short courses of narrowspectrum parenteral antimicrobials are justified. In studies of the etiology of
childhood consolidated pneumonia, lung taps provide specific data with a
relatively low rate of adverse events.

11

2 ABBREVIATIONS
CF

complement fixation

Cfm

cefuroxime

C-PS

C-polysaccharide

CRP

C-reactive protein

EIA

enzyme immunoassay

ESR

erythrocyte sedimentation rate

Hib

Haemophilus influenzae type b

Ig

immunoglobulin

IC

immune complex

i.m.

intramuscularly

i.v.

intravenously

MIC

minimal inhibitory concentration

MIF

micro-IF, microimmunofluorescence

NPA

nasopharyngeal aspirate

PCR

polymerase chain reaction

Pen

procaine penicillin

Ply

pneumolysin

RSV

respiratory syncytial virus

WBC

white blood cell count

12

3 LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following articles, which are referred to in the text
with roman numerals (I-V).
I

Vuori E, Peltola H, Kallio MJT, Leinonen M, Hedman K, SE-TU Study

Group. Etiology of pneumonia and other common childhood infections
requiring hospitalization and parenteral antimicrobial therapy. Clin Infect
Dis 1998;27:566-572.

II Vuori-Holopainen E, Peltola H, Kallio MJT, SE-TU Study Group. Narrowversus broad-spectrum parenteral antimicrobials against common infections
of childhood: a prospective and randomised comparison between penicillin
and cefuroxime. Eur J Pediatr 2000;159:878-884.
III Peltola H, Vuori-Holopainen E, Kallio MJT, SE-TU Study Group. Successful
shortening from seven to four days of parenteral beta-lactam treatment for
common childhood infections: A prospective and randomized study. Int J
Infect Dis 2001;5:3-8.
IV Vuori-Holopainen E, Salo E, Saxn H, Vaara M, Tarkka E, Peltola H. Clinical
pneumococcal pneumonia due to Moraxella osloensis: Case report and a
review. Scand J Infect Dis 2001;33:625-627.
V Vuori-Holopainen E, Salo E, Saxn H, Hedman K, Hyypi T, Lahdenper R,
Leinonen M, Tarkka E, Vaara M, Peltola H. Etiological diagnosis of
childhood pneumonia by lung tap, applying modern microbiology. Submitted.
The thesis also includes some previously unpublished data.

13

4 INTRODUCTION
In nonindustrialized countries, the most frequent causes of morbidity and
mortality among children are infectious diseases, especially lower respiratory
infections (Berman 1991, Pechre 1995). Annually, about three million children
die from pneumonia (Berman 1991). In industrialized countries, lower respiratory
infections are also a significant cause of morbidity and hospitalization in children
< 5 years of age (Murphy et al. 1981, Jokinen et al. 1993, Ruuskanen and
Mertsola 1999).
Despite the frequency of these diseases, the causes of pneumonia and other
common invasive bacterial infections are seldom identified in clinical practice.
In Europe, knowledge of the etiology of pneumonia has mostly been based on
serology and antigen detection methods (Ruuskanen and Mertsola 1999). With
few exceptions, serology requires paired serum samples, and the test results are
only available after several weeks. Culture-based methods are more rapid, but
with the exception of blood cultures, which are positive in only 0 to 5 % of
children requiring hospitalization (Nohynek et al. 1991, Clements and Stephenson
1996, Genrel et al. 1997, Juvn et al. 2000), they are frequently complicated by
the difficulty of obtaining accurate specimens. Bacteria detected in upper airway
samples only demonstrate carriage, and often have little or no association with
the infection in the lower airways (Cunanan et al. 1979, Korppi et al. 1992b). In
nonbacteremic cases, convincing evidence of etiology is obtained only by
identification of an agent from the pleural fluid or lung parenchyma (Finland
1969, Ruuskanen and Mertsola 1999).
The low diagnostic yield has contributed to the frequent and inappropriate
use of broad-spectrum antimicrobials in childhood infections (Clements et al.
2000). However, the use of antimicrobials should be restricted, since the amounts
prescribed, their antimicrobial spectra, and the lengths of treatment are strong
predictors of the development and spread of resistant micro-organisms (Arason
et al. 1996, Goldmann et al. 1996, Seppl et al. 1997, Swartz 1997, Guillemot
et al. 1998). Consequently, in addition to etiologic data, clinical trials of the
efficacy of an antimicrobial regimen are needed to establish recommendations
regarding appropriate treatment in different clinical situations (Berner 2000).
In the present study, the effectiveness of two antimicrobial regimens and two
different lengths of treatment were compared in a prospective, randomized series
of children with signs and symptoms suggestive of an acute invasive bacterial
infection. The etiology was investigated both by conventional methods and by
serology. In a further study of childhood community-acquired pneumonia, another
approach was taken. Lung tap (transthoracic needle aspiration) was performed
on hospitalized patients with consolidated pneumonia. The use and limitations
of the lung tap were elucidated by comparing the tap yields with those obtained
by other routine methods.

14

5 REVIEW OF THE LITERATURE

5.1. Definition of lower respiratory infections
Pneumonia is defined as inflammation and consolidation of lung tissue due to an
infectious agent (Marrie 1994, Jadavji et al. 1997). The clinical symptoms and
signs are nonspecific and variable (Jadavji et al. 1997, Tan et al. 1998), and
therefore, in industrialized countries, radiologic confirmation of pneumonia is
considered the gold standard (Marrie 1994, Bartlett and Mundy 1995, Jadavji
et al. 1997). In nonindustrialized countries, radiographic facilities are so seldom
available that the World Health Organization defines moderate to severe pneumonia
in those circumstances as an illness with cough or difficulty in breathing, increased
respiratory rate and chest indrawing (WHO 1994, Shann 1995).
In community-acquired pneumonia, the infection has been acquired outside
the hospital, and there are two main classic presentations (Jadavji et al. 1997):
1) typical pneumonia with acute onset, dominated by symptoms of fever, chills,
pleuritic chest pain, and a productive cough, and 2) atypical pneumonia with
gradual onset over several days to weeks, dominated by symptoms of headache
and malaise, nonproductive cough, and low-grade fever. However, in many
cases the clinical manifestations differ from these classic presentations, and
the etiologic agent cannot be identified on the basis of the clinical symptoms
and signs.
Radiographically, pneumonia has been divided traditionally into two
manifestations: interstitial and alveolar. Diffuse interstitial infiltrates tend to be
seen in viral infections (Jadavji et al. 1997), while lobar consolidation suggests
a bacterial etiology. Alveolar infiltrates are seen in both bacterial and viral
pneumonia (Jadavji et al. 1997). On chest radiograph, the size and shape of the
infiltration change during the course of infection and influence the radiographic
signs. The value of chest radiographs in differentiating bacterial from viral lower
respiratory infections in children was recently reviewed (Swingler 2000), and
no clinically relevant accuracy was demonstrated.

5.2. Epidemiology and pathogenesis of lower

respiratory infections
5.2.1. Epidemiology
The overall incidence of acute respiratory infections is very similar in industrialized
and in nonindustrialized countries (Berman and McIntosh 1985), but there are
marked differences in their severity. The incidence of lower respiratory infections in

15
the nonindustrialized world is as much as 10 times higher than in industrialized
countries, being in some areas 300/1 000/year among children < 5 years of
age (McCracken 2000). In the Finnish population, in contrast, the overall
incidence of community-acquired pneumonia is 37/1 000/year among children
< 5 years of age, and 16/1 000/year among those between 5 and 14 years
(Jokinen et al. 1993). In the USA, the incidence of pneumonia in children < 6
years of age was 30/1 000/year in 1966-1971 (Foy et al. 1973). In general,
the incidence varies with the social status, nutritional state, passive exposure
to smoke, and attendance at a day-care center (Jadavji et al. 1997). However,
in a recent Finnish population based analysis the most important risk factor
for community-acquired pneumonia in children was susceptibility to respiratory
infections (Heiskanen-Kosma et al. 1997).
Not only the incidence, but also the mortality from pneumonia is higher in
the developing countries, mostly on account of the greater severity of the disease.
Worldwide, pneumonia is responsible for a quarter of all deaths in children < 5
years of age (Berman 1991). In industrialized countries, the mortality rate has
declined markedly in recent decades (Dowell et al. 2000). In the USA, the case
fatality of pneumonia in children has declined by 97 %, from 24,637 cases in
1939 to 800 in 1996 (Dowell et al. 2000). In Finland, three children died from
community-acquired pneumonia in 1994-1995 (Ruuskanen and Mertsola 1999).
In our circumstances, approximately half of the < 5-year-old children with
pneumonia are admitted to hospital, whereas most of the older children are
treated at home (Jokinen et al. 1993). In future, vaccines against S. pneumoniae
and influenza virus may offer a chance to reduce the incidence of pneumonia in
both the industrialized and the nonindustrialized world (King et al. 1998, Rubin
2000), as has already occurred for Haemophilus influenzae type b (Hib)
pneumonia in the industrialized countries as a result of the introduction of general
vaccinations (Peltola 2000).

5.2.2. Pathogenesis
Bacteria causing pneumonia may gain access to the lung by aspiration of mucus
contaminated by the normal oropharyngeal flora. The pathogens ability to adhere
to the respiratory tract epithelium and to overcome the hosts defense mechanisms
is crucial for infectivity (Bakaletz 1995). In most cases, adhesion depends on
epithelial damage, which may be caused by a concomitant or previous upper
respiratory viral infection (Fainstein et al. 1980). In addition to damaging the
mucosal cells, viruses lead to pulmonary infection by causing increased mucus
production, decreased ciliary action, epithelial swelling and dysfunction of
leukocytes (Berendt et al. 1975, Carson et al. 1985).

16
In acute otitis media, evidence for interactions between bacteria and viruses
has been obtained from both epidemiologic and microbiologic studies
(Hendersson et al. 1982, Ruuskanen et al. 1989). The peak incidence of the
viral illness occurs either concurrently with or immediately preceding that of
the bacterial disease (Ruuskanen et al. 1989). Viral infections in the nasopharynx
seem to relate more directly to the development of otitis media than
nasopharyngeal colonization with S. pneumoniae or H. influenzae alone
(Hendersson et al. 1982). In children with invasive pneumococcal disease, the
association with viral respiratory infection is deemed less apparent and less
immediate, the lag period being approximately four weeks (Kim et al. 1996).
However, previous influenza infection has been demonstrated to predispose to
severe pneumococcal pneumonia (OBrien et al. 2000).
The outcome of infection depends on the balance between the host defense
mechanisms and the growth rate of the invading pathogen (Johnston 1991). In the
lungs, alveolar macrophages and polymorphonuclear leukocytes clear the bacteria,
provided that specific antibodies and complement are present. In the absence of
specific antibodies, clearance of bacteria may be enhanced by C-reactive protein
(CRP) -mediated complement activation (Johnston 1991, Jarva et al. 1999).

5.3. Pathogens associated with lower

respiratory infections
A wide range of pathogens has been associated with lower respiratory infections.
The spectrum of etiologic agents in industrialized countries resembles that in
nonindustrialized countries (Table 1), albeit pathogens such as Staphylococcus
aureus account for a higher proportion of cases in developing countries. In the
industrialized world, viruses cause a high proportion of cases. According to the
culture-based studies, the predominant bacteria causing childhood lower
respiratory infections worldwide are pneumococci along with H. influenzae
(Klein 1981, Shann 1986, Vuori-Holopainen and Peltola 2001). However, the
etiology of pneumonia is a complex issue, and many cases remain without an
identified etiology. Some series from nonindustrialized countries have reported
up to 15-45 % of blood cultures as positive (Fagbule 1993, Adegbola et al.
1994), whereas, in industrialized countries, positive results are obtained in only
approximately 0-5 % of cases (Paisley et al. 1984, Nohynek et al. 1991, Genrel
et al. 1997, Juvn et al. 2000). Sometimes, different organisms have been detected
simultaneously from blood and lung (Shann et al. 1984, Falade et al. 1997); in
fact, bacteremia has been suggested to be secondary to initially non-bacteremic
pneumonia (Scott and Hall 1999). This may explain the higher incidence of
positive blood cultures in nonindustrialized countries.

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Table 1. Overall frequency of the bacterial agents in studies of pneumonia in children
conducted since 1980.
Lung tap studies
Continent Lung tap S. pneumoniae H. influenzae M. catarrhalis S. aureus E. coli
N
N %
N
%
N
%
N %
N %

Klebsiella spp Others

N
%
N %

Reference

Africa

462

61

13

13

0.2

83 18

17

55

12

44 10

Vuori-Holopainen
et al. 2001

Asia

292

29

10

1.4

0.3

40 14

12

36 12

Vuori-Holopainen
et al. 2001

Oceania

101

34

34

42

42

23 23

Vuori-Holopainen
et al. 2001

Serology-based studies*
Continent Patients S. pneumoniae H. influenzae M. catarrhalis S. aureus E. coli

Klebsiella spp Others

Africa

164

14

9**

13

ND***

ND

ND

15

Asia

100

ND

ND

ND

30 30

Forgie et al.
1991a, 1991b
Yang et al. 2000

North
America

168

35

27

ND

ND

ND

ND

22 13

Wubbel et al.1999

Europe

1157

244

21

76

ND

ND

ND

118 10

Claesson et al.1989,
Ruuskanen et. al.
1992, Korppi et al.
1993a, Nohynek
et al. 1995,
Heiskanen-Kosma
et al.1998,
Juven et al. 2000

ND

31

Only some of the representative studies included. If several methods were used, only serologic results were accepted.

**

Percentage counted from patients in whom the specific agent was searched for by serology.

***

No data

According to a recent review of lung tap in childhood pneumonia, a pathogen was

identified in 32 % to 66 % of patients from different continents, even though in
most cases standard culture techniques only were used (Vuori-Holopainen and
Peltola 2001). However, very few data were available from the western world.
Most etiologic information from developed countries has been derived either from
retrospective analyses or from studies of hospitalized patients. The results of such
studies are difficult to compare because of different inclusion criteria and diagnostic
methods. Table 2 presents a summary of etiologic studies from developed countries.

18
Table 2. Studies from developed countries in which both viral and bacterial
etiology (%) in childhood pneumonia were searched for.
Country

% of patients
hospitalized

No of microbes
studied

Etiology
detected

Bacterial

Mixed viralbacterial

Viral

USA

102

100

16

85

11

28

47

USA

162

20

17

48

22

19

USA

98

21

10

48

10

39

USA

168

13

43

25

15

France

104

50

12

84

47

29

Spain

198

19

11

79

58**

Sweden

336

50

13

48

19

25

Finland

121

100

15

70

25

20

25

Finland

50

100

16

88

28

34

26

Finland

195

100

14

50

15

16

19

Finland

201

32

13

66

41

10

15

Finland

254

100

15

85

22

30

32

66

22

16

27

mean %

ND

27**

* Pnc, S. pneumoniae; Hi, nontypable H. influenzae; Hib, H. influenzae type b; M. cat, M. catarrhalis; M. pn, M. pneumoniae.
** Two pathogens from 11 children, not specified whether two bacteria, two viruses, or mixed viral-bacterial infections.

19
Table 2. Continued.

Country

Pnc* Hi/Hib* M.cat* M. pn* RSV

Adeno Influenza Parainfluenza Rhino

virus A or B
1, 2, or 3
virus

Reference

USA

102

23

2/3

ND

ND

60

Paisley et al. 1984

USA

162

18

ND/7

ND

Ramsey et al. 1986

USA

98

17

ND/2

ND

28

ND

Turner et al. 1987

USA

168

27 ND/ND

ND

ND

Wubbel et al. 1999

France 104

13 ND/ND

ND

41

10

ND

Genrlel et al. 1997

Spain

198

15

ND/2

ND

40

15

ND

Ausina et al. 1988

Sweden 336

13

ND/1

ND

10

20

ND

Finland 121

16

16/2

28

13

ND

Claesson et al.
1989
Nohynek et al.1991

Finland

50

38

12/ND

10

20

30

10

10

Finland 195

21

21/ND

27

ND

ND

Finland 201

28

6/ND

22

21

ND

Finland 254

37

9/ND

29

10

24

mean %

22

11/3

16

23

11

Ruuskanen
et al.1992
Korppi et al. 1993a
Heiskanen-Kosma
et al.1998
Juvn et al. 2000

* Pnc, S. pneumoniae; Hi, nontypable H. influenzae; Hib, H. influenzae type b; M. cat, M. catarrhalis; M. pn, M. pneumoniae.
** Two pathogens from 11 children, not specified whether two bacteria, two viruses, or mixed viral-bacterial infections.

20

5.3.1. Streptococcus pneumoniae

5.3.1.1. Serotypes and virulence factors
S. pneumoniae is enveloped by a polysaccharide capsule determining the serotype
of each pneumococcus. According to the Danish nomenclature (Forward 1999),
90 serotypes have been recognized to date. Based on information obtained mostly
from otitis media, meningitis, and blood cultures, the serogroups currently
considered to be the most important among pediatric patients in industrialized
countries are 1, 4, 6, 7, 9, 14, 15, 18, 19, and 23 (Eskola et al. 1992, Butler et
al. 1995, Sniadack et al. 1995, Toikka et al. 1999b). Recently, in a Finnish
analysis of otitis media most common serogroups were found to be 6, 11, 15,
18, 19, and 23 (Eskola et al. 2001). Since childhood pneumonia is rarely
bacteremic and lung aspirates are seldom obtained, only limited data on
pneumococcal serogroups in pneumonia are available (Table 3).
Besides capsular polysaccharides, pneumococci have several other virulence
factors, such as C-polysaccharides (C-PS) and pneumolysin (Ply), a toxic
intracellular protein, both of which are common to all strains (Tuomanen et al.
1987, Paton et al. 1993). Pneumococcal surface protein A and surface adhesin
A are also found in most clinical isolates, and these components, too, may
contribute to the pathogenicity of S. pneumoniae (Briles et al. 1988, Rapola et
al. 2000). In pneumococcal infections, antibody responses may be detected to
all these virulence factors.

Table 3. Pneumococcal serogroups recovered from childhood pneumonia.

Source of
isolates*

Number of
isolates

Country

Age

Serogroups**

Reference

Papua New Guinea

ND***

5, 6, 7, 14, 16, 46

Riley et al. 1983

LA, B

25

Papua New Guinea

1m-4y

6, 14, 19, 5, 16, 23, 4, 12, 15, 17, 22, 25, 45

Shann et al.1984

LA, B, PF

46

Gambia

3m-5y

6, 14, 5, 1, 9, 12, 13, 19

Adegbola et al.
1994

LA, B

14

Gambia

2m-10y

1, 5, 6, 14, 19

ONeill et al.
1989

61

Finland

15y

7, 19, 14, 6, 23, 9, 4, 3, 10, 38

Toikka et al.
1999b

LA

LA, lung aspirate; B, blood; PF, pleural fluid.

** Presented in descending order of prevalence; numbers of serotypes found in >10 % of isolates bolded.
*** ND, no data.

21
5.3.1.2. Carriage
Pneumococci colonize the human pharynx commonly and asymptomatically;
> 95 % of children carry pneumococcus at some time during the first 2 years of
life (Klugman 1990). The way of potential translocation of pneumococci from
the nasopharynx to the middle ear, lungs, blood, or meninges is not well
understood. In most cases, symptomatic infection arises from a recently acquired
serotype (Gray et al. 1980). Acquisition of a new serotype leads to infection in
15 % of cases, and approximately 75 % of pneumococcal infections occur within
one month after acquisition (Gray et al. 1980). In 90 % of children with an invasive
disease, pneumococcus has been isolated from the nasopharynx, the strains
belonging to the same serotype in 77 % of cases (Lloyd-Evans et al. 1996).
5.3.1.3. Pneumococcal infections
S. pneumoniae is considered to be the most frequent bacterial cause of
pneumonia, otitis media, sinusitis, and bacteremia. In areas where Hib has been
virtually eliminated by vaccine, it is also often the main pathogen in childhood
meningitis (Klein 1981, Musher 1992). According to serology-based studies,
pneumococcus is, with few exceptions (Yang et al. 2000), the most common
causative agent of childhood pneumonia in both industrialized and nonindustrialized countries (Tables 1 and 2). In North America and in Europe, it is
found in 18-27 % and 13-38 % of children with mainly hospitalized pneumonia,
respectively. The full role of pneumococcus is difficult to assess, since all available
assays for optimal detection of pneumococci have seldom been used (Heiskanen-Kosma 2000). In lung tap studies on 855 pediatric pneumonia patients
conducted since 1980, a pneumococcal etiology has been confirmed in only 10 %
of Asian, 13 % of African, and 34 % of Oceanian children (Table 1). In the older
literature from Europe and North America pneumococcus was reported in 32 %
(81/255) and 10 % (63/645) of lung aspirates, respectively (Vuori-Holopainen
and Peltola 2001). However, the results may have been influenced, especially in
the developing countries, by poor methodology (Scott and Hall 1999).
Some lung tap-based studies have related the pneumococcal etiology to the
lobar and bronchopneumonia. S. pneumoniae was found in 78 % of patients
with lobar pneumonia, in contrast to only 13 % of those with bronchopneumonia
(Vuori-Holopainen and Peltola 2001).
The incidence of pneumococcal disease is highest in young infants, with a
predominance of males in all clinical manifestations (Klein 1981, Toikka et al.
1999b). Among hospitalized children, about one-third have pneumonia and onethird a nonpulmonary infection such as meningitis or acute otitis media, the rest

22
having no identifiable focuses (Klein 1981). The peak incidence of hospitalization
for pneumococcal pneumonia has been reported among infants from 13 to 18
months of age (Klein 1981), but in a recent Finnish analysis of bacteremic
pneumococcal pneumonia (N 85) the mean age was higher, 3.6 years (Toikka et
al. 1999b).
5.3.1.4. Prevention of pneumococcal infection
The steady increase in pneumococcal resistance to several antimicrobials has
prompted prevention of pneumococcal infections by immunization.
Polysaccharide vaccines consisting of 23 capsular types are available, but they
are poorly immunogenic in infants and young children (Mkel et al. 1983,
Rubin 2000). Therefore, conjugate vaccines have recently been developed. They
have improved immunogenicity, and are more effective in eradicating
pneumococci from the nasopharynx (Rubin 2000). A heptavalent vaccine is
estimated to be able to prevent up to 86 % of pneumococcal bacteremias and 83 %
of pneumococcal meningitides in the USA (Butler et al. 1995). Recently, it has
been shown to reduce the number of pneumococcal otitis media episodes by 33 %
(Eskola et al. 2001). The potential effectiveness against pneumonia is less well
known, although recent preliminary results from the USA suggest a reduction
of up to 35 % in the incidence of radiologically proven childhood pneumonia
(Shinefield and Black 2000).

5.3.2. Haemophilus influenzae

Six encapsulated and several nonencapsulated (nontypable) strains of H.
influenzae have been identified. The most pathogenic is type b, which causes 5 to
18 % of cases of bacterial pneumonia in unvaccinated areas (Jadavji et al. 1997).
The virulence of nontypable strains is lower, and asymptomatic children frequently
carry these in their nasopharynges (Hjuler et al. 1995).
Nontypable H. influenzae is commonly found in patients with upper
respiratory infection or acute otitis media, and is probably also an important
cause of lower respiratory infections. Serologically verified nontypable H.
influenzae infection is found in 6 to 17 % of children with community-acquired
pneumonia (Nohynek et al. 1991, Ruuskanen et al. 1992, Korppi et al. 1993a,
Heiskanen-Kosma et al. 1998, Juvn et al. 2000). Up to 80 % of H. influenzae
infections have been reported to occur as mixed infections with a virus or as
dual infections with another bacterium (Korppi et al. 1993a). Convincing
evidence for the pathogenicity of nontypable strains has been obtained from
bronchoalveolar lavage (Wang et al. 1994) and lung aspirate cultures (Shann et
al. 1984). According to lung tap studies, H. influenzae is found more often in

23
bronchopneumonia than in lobar pneumonia (20 % vs. 7 %) (Vuori-Holopainen
and Peltola 2001).
The highest frequency of culture-positive H. influenzae pneumonia has been
reported from Papua New Guinea, where this agent was found in 80 % of lung
aspirates, only 12 % being of type b (Shann et al. 1984). In contrast, in Africa
and Asia, H. influenzae has been isolated only in 3 % and 1.4 % of lung aspirates,
respectively (Table 1). These great differences may be explained by suboptimal
methods for detecting H. influenzae (Scott and Hall 1999).

5.3.3. Moraxella catarrhalis

M. catarrhalis is an important cause of otitis media and sinusitis, but its role in
lower respiratory infections is less clear. Serologic evidence for M. catarrhalis
has been found in 2 to 10 % of hospitalized children with pneumonia (Nohynek
et al. 1991, Ruuskanen et al. 1992, Korppi et al. 1993a, Heiskanen-Kosma et
al. 1998, Juvn et al. 2000), but few studies have reported it from lung aspirates
(Abdel-Khalik et al. 1938, Shann et al. 1984, Wall et al. 1986). Notably, M.
catarrhalis often appears as a co-pathogen in both lung tap studies (AbdelKhalik et al. 1938, Shann et al. 1984) and serology-based studies (Nohynek et
al. 1991, Korppi et al. 1992a, Korppi et al. 1993a). This suggests that it is
rarely a primary cause of pneumonia in children.

5.3.4. Mycoplasma pneumoniae

M. pneumoniae and Chlamydia pneumoniae are naturally resistant to -lactam
antimicrobials. These bacteria cause epidemics at intervals of a few years
(Hammerschlag 1995, Heiskanen-Kosma et al. 1999). Naso- and oropharyngeal
carriage is common in asymptomatic children, especially during epidemic periods
(McMillan et al. 1986, Foy 1993, Normann et al. 1998), and after symptomatic
infection, the carriage of M. pneumoniae may last for months (Foy 1993). Thus,
detection of M. pneumoniae in the throat of a school-aged child does not imply
that the current disease is caused by this organism (McMillan et al. 1986).
Mycoplasmal infections are common in children > 5 years of age, the incidence
being highest at school age (Foy 1993). However, some recent data suggest its
importance in younger children also (Hammerschlag 1995). Most often M.
pneumoniae causes mild upper respiratory infections such as sore throat,
pharyngitis, and tracheitis, with tracheobronchitis or pneumonia developing in
only 5 to 10 % of patients (Wendelien Dorigo-Zetsma et al. 2001). Probably
because of its relative mildness, mycoplasmal pneumonia is found more frequently
in ambulatory (20 to 40 %) than in hospitalized patients (10 to 20 %) (Foy

24
1993, Korppi et al. 1993a, Heiskanen-Kosma et al. 1998, Ruuskanen and
Mertsola 1999). Mycoplasma diagnostics are usually based either on
measurement of serologic responses or on polymerase chain reaction (PCR)
from naso- or oropharyngeal aspirates, but isolation of mycoplasma from
pleural fluid provides convincing evidence for its pathogenic role in pediatric
patients, too (Loo et al. 1991).

5.3.5. Chlamydia pneumoniae

Serologic surveys have shown a rising prevalence of C. pneumoniae antibodies
with increasing age. Antibodies are found in 10 % of children from 5 to 10 years
of age and in 30 to 45 % in adolescence (Grayston et al. 1990). However, most
infections are mild or asymptomatic; clinical pneumonia occurs in only 10 %
(Jadavji et al. 1997). In the USA, C. pneumoniae has been reported to be
responsible for 6 % of community-acquired cases of pneumonia in ambulatory
children (Wubbel et al. 1999). In contrast, in a Finnish population-based study
comprising both ambulatory and hospitalized patients, C. pneumoniae was found
in 9 % of children between 5 and 9 years of age and in 31 % of those 10 years
of age (Heiskanen-Kosma et al. 1999). In another Finnish series including only
hospitalized children, C. pneumoniae was detected in 3 % of cases (Juvn et al.
2000), suggesting that most C. pneumoniae infections are mild. C. trachomatis
may also cause respiratory infections sometimes, especially in young infants in
the USA and in developing countries (Hammerschlag 1994).

5.3.6. Respiratory viruses

A wide range of viruses, such as RS, rhino-, parainfluenza, influenza, adeno-,
corona-, entero-, varicella-zoster-, and human herpes virus type 6 (HHV-6),
have been associated with childhood pneumonia (Jadavji et al. 1997, Ruuskanen and Mertsola 1999). Overall, they have been estimated to account for 20 to
62 % of cases, being found especially frequently in children < 2 years of age
(Jadavji et al. 1997, Ruuskanen and Mertsola 1999, Wubbel et al. 1999, Juvn
et al. 2000). RS virus is detected most often in small children, especially in
infants (Korppi et al. 1993a). It was found in 30 to 45 % of pneumonia cases
during an epidemic compared to 5 % in the non-epidemic season (Juvn et al.
2000). Rhinoviruses are important causes of the common cold (Mkel et al.
1998), but recently they have also been found in 24 % of nasopharyngeal aspirates
(NPA) of children hospitalized for pneumonia (Juvn et al. 2000). Some further

25
evidence for the role of rhinoviruses in lower respiratory infections has been
obtained from studies on bronchial samples and post-mortem lung biopsies
(Schmidt et al. 1991, Imakita et al. 2000).
In general, it is not known whether a virus isolated from the upper respiratory
tract is correlated with infection in the lower respiratory tract, or has only paved
the way for a bacterial pathogen. Hughes et al. compared virus isolations from
the upper respiratory tract with those from lung aspirates. In two out of eight
cases, the same virus (parainfluenza type 2, polio type 1) was found in both
samples. In six cases, a virus was isolated only from the upper respiratory tract,
but never only from the lung (Hughes et al. 1969). In a post-mortem study of
13 lung tissue samples, viruses were isolated from the lung in six cases, three of
which were also found in the NPA (Avila et al. 1990). Viral diagnostics have
been performed from lung aspirates obtained from consolidated pneumonia in
at least nine studies (Vuori-Holopainen and Peltola 2001), the total identification rate being only 4 %. With the exception of a study from The Gambia, in
which viruses were isolated in 30 % (28/94) of aspirates (Adegbola et al.
1994), the virologic yield from lung aspirates has been very low. This low
yield may partly be explained by technical problems and lack of modern
virologic methods.

5.3.7. Mixed infections

Several, mostly serology-based, studies suggest that mixed viral-bacterial
infections are common, being found in 10 to 34 % of childhood pneumonias
(Table 2). Bacterial coinfection has been detected with the greatest frequency in
respiratory syncytial virus (RSV) infections (Paisley et al. 1984, Turner et al.
1987, Korppi et al. 1989, Korppi et al. 1993a). Recently evidence for concomitant
bacterial infection was reported also in nearly half of patients with rhino-,
parainfluenza -, or adenovirus associated pneumonia (Juvn et al. 2000).
Dual bacterial infections have also been detected in both serologic (Nohynek
et al. 1991, Heiskanen-Kosma et al. 1998, Juvn et al. 2000) and lung aspirate
studies (Shann et al. 1984). A multiple bacterial etiology appears to be more
common in children than in adults (Scott and Hall 1999). Besides being caused
by two different species, dual infection may also occur within a single species.
Two serotypes of pneumococci have been isolated simultaneously from lung
and blood samples (Falade et al. 1997), and two different populations of
H. influenzae from lung aspirates (Shann et al. 1984).

26

5.4. Methods for identification of etiology

A wide range of microbiologic methods based on a variety of patient samples
has been developed to identify the etiology of pneumonia and other invasive
infections. In pneumonia, blood, serum, urine, throat swab, NPA, bronchoalveolar
lavage, lung biopsy, pleural fluid and lung aspirate have been used. Involvement
of a pathogen has been shown directly by culture, antigen detection, or nucleic
acid detection, or by measuring specific antibodies or immune complexes.
However, in obtaining valid information, several problems have emerged. Many
methods, especially in bacterial infections, have proven unreliable in terms of
either low sensitivity or specificity, or the difficulty of obtaining representative
samples (Table 4). Cover of all potential pathogens requires a wide, expensive,
and laborious panel of methods.

Table 4. Problems posed by different microbiologic methods in the search for

bacterial etiology in pneumonia.
Method

Problem

Culture
Blood

Low sensitivity

Sputum

Contamination with oropharyngeal flora

Oropharyngeal specimen

High carriage rate of potential pathogenic bacteria

Bronchoalveolar lavage

Requires anesthesia in children

Lung aspirate

Invasive method, risk of pneumothorax

Other
Antigen detection

Disputable sensitivity and specificity, depending on samples

Antibody assay

Paired samples usually required, disputable sensitivity and specificity

Immune complex detection

Disputable sensitivity and specificity

Nucleic acid detection

Varying sensitivity, depending on samples

27

5.4.1. Bacterial agents

5.4.1.1. Culture
A positive culture from a normally sterile body site - in pneumonia from blood,
pleural fluid, transtracheal aspirate, or lung aspirate is considered the most
specific indicator for microbiologic diagnosis of bacterial infection (Ruuskanen
and Mertsola 1999). Studies of bacteremic diseases, however, indicate that
blood culture has a very low sensitivity (Isaacman et al. 1996). Negative results,
especially in children, may reflect intermittent and low-density ( 10 to 15
organisms per milliliter) bacteremia (Isaacman et al. 1996). Collecting a single large-volume blood sample may slightly increase the sensitivity (Isaacman
et al. 1996).
Gram staining and culture of a well-produced sputum sample may be
diagnostic in adults and in adolescents (Jadavji et al. 1997), but in children the
sample is frequently contaminated with upper respiratory tract pathogens (Korppi
et al. 1992b). For the same reason, naso- and oropharyngeal specimens cannot
be used (Klein 1981, Foy 1993, Normann et al. 1998).
Since, moreover, pleural fluid is seldom present in uncomplicated pneumonia
(Prez et al. 2001), invasive diagnostic techniques such as transtracheal aspiration,
bronchoscopy with a protected brush catheter, bronchoalveolar lavage, and lung
tap have been used. In clinical practice, however, their use has been restricted
by risk of potential adverse events.
5.4.1.2. Antigen detection
Soluble antigenic components of bacteria may be detected in a sample of
concentrated urine, serum, sputum, pleural fluid, or lung aspirate (Farrington
and Rubenstein 1991, Boersma et al. 1991). Commercial agglutination reagents
are available for pneumococcus and Hib, but not for the other serotypes of
H. influenzae. Unfortunately, most assays have limited specificity. False-positive
results may be obtained because of cross-reactions with other bacteria or mucosal
damage caused only by viruses, and serum or urine may be positive because of
pneumococcal infection at sites other than the lungs (Ramsey et al. 1986,
Bromberg and Hammerschlag 1987, ONeill et al. 1989, Korppi et al. 1993b).
Furthermore, polysaccharide excretion may continue for several weeks after
acute infection or vaccination (Bromberg and Hammerschlag 1987, Kelley and
Keyserling 1997). In respiratory secretions, it may be difficult to distinguish
infection from colonization (Korppi et al. 1992b). False-negative results from

28
serum samples are also relatively common (Silvermann et al. 1977, ONeill et
al. 1989). Consequently, despite the rapidity and technical simplicity of antigen
detection assays, it may be doubted whether they are reliable diagnostic methods
in pneumonia.
5.4.1.3. Antibody and immune complex assays
Measurements of antibody responses and microbe-specific immune complexes
(IC) from serum samples have been used in the etiologic diagnostics of lower
respiratory infections in both adults (Lehtomki et al. 1988, Jalonen et al. 1989,
Lieberman et al. 1996) and children (Nohynek et al. 1995, Heiskanen-Kosma et
al. 1998, Korppi and Leinonen 1997, 1998, Juvn et al. 2000).
A bacterial antibody response as such, may indicate various host-pathogen
relationships: carriage, carriage acquisition, subclinical infection, or clinical
disease (Gwaltney et al. 1975, Nohynek et al. 1995). Furthermore, several
concomitant clinical manifestations such as acute otitis media may cause the
positive serologic response (Leinonen et al. 1981, Virolainen et al. 1996, Rapola et al. 2001). On the other hand, viral infection does not often seem to cause
seroresponse to bacterial agents. Only two of 200 young adults with common
cold developed positive antibody response to either S. pneumoniae or to
H. influenzae (Mkel et al. 1998). Furthermore, in a study of 52 children with
apparent viral laryngitis, only three had a positive antibody response to a
respiratory bacterium (Korppi et al. 1990). In a series of 200 healthy adults and
children, a diagnostic rise for S. pneumoniae, H. influenzae or M. catarrhalis
was detected in less than 1 % of paired sera (Korppi et al. 1989). The sensitivity
and specificity of these assays in childhood pneumonia cannot be determined
precisely, since serology has only rarely been compared with culture of bacteria
isolated from blood (Toikka et al. 1999a) and never with bacteria isolated from
an infected lung.
5.4.1.3.1. Pneumococcal serology
Pneumococcal infection induces serum antibody responses to Ply, to C-PS, and
to type-specific capsular polysaccharides, the first two being common to all
pneumococcal strains. The enzyme immunoassay (EIA) for measuring IgG
antibodies to Ply seems to be a useful tool for the diagnosis of pneumococcal
pneumonia in both adults (Jalonen et al. 1989, Lieberman et al. 1996) and children
(Korppi et al. 1992b, Nohynek et al. 1995), although the sensitivity of the assay
is lower in children (Claesson et al. 1989). False positive results in children are
considered rare; a diagnostic increase in titer was demonstrated in only 1 to 2.7 %
of serum samples from healthy children (Korppi et al. 1993a, Nohynek et al.
1995). The presence of otitis media or pneumococcal carriage was not associated

29
with any higher frequency of antibody response to pneumolysin in 121 children
with acute lower respiratory infection (Nohynek et al. 1995). In children with
verified pneumococcal otitis media, IgA antibodies to Ply were usually found
(Virolainen et al. 1996). Recently, however, it was shown that IgG antibodies
to Ply are also induced by carriage and by pneumococcal otitis media (Rapola
et al. 2001).
Measurement of type-specific capsular polysaccharides has proven sensitive
in adults, but, in children < 2 years of age, antibody formation is weak (Claesson
et al. 1989). In children, measurement of C-PS antibodies has also been used in
the serologic diagnosis of pneumococcal infections (Koskela 1987) including
pneumonia (Korppi et al. 1993b, Wubbel et al. 1999, Juvn et al. 2000). False
positive results in healthy children have been rare (Korppi et al. 1989).
In many invasive infections, including those caused by S. pneumoniae, antigens
and antibodies are partly sequestered in ICs in the circulation (Korppi and Leinonen 1997, 1998). In assays measuring free antibodies and in antigen detection
tests, this may result in false-negative results. Methods for detecting
pneumococcal ICs from either acute or convalescent serum samples have been
developed (Mellencamp et al. 1987, Leinonen et al. 1990, Holloway et al. 1993).
Measurements of ICs specific to Ply and C-PS have been found to be more
sensitive and specific than conventional antibody assays (Korppi and Leinonen 1997, 1998). In infants, however, measurement of circulating ICs has been
as insensitive as other antibody assays (Korppi and Leinonen 1998). Furthermore,
recently in a study comprising adults, Ply-ICs were shown to be present in acutephase serum samples from healthy pneumococcal carriers more often than in
those from patients with bacteremic pneumococcal pneumonia (Musher et al.
2001). In studies in which several serologic tests have been used for pneumococcal
diagnostics, often only one test has been positive (Korppi and Leinonen 1998,
Heiskanen-Kosma 2000, Toikka et al. 2000). This suggests limited sensitivity
of all the currently available tests.
5.4.1.3.2. Other bacterial serology
In contrast to pneumococcal strains, no tests for detecting antibodies against
antigens common to all H. influenzae or all M. catarrhalis strains have been
commonly available. Serum antibodies to these bacteria can be measured by
EIA, with a mixture of whole cell antigens of 10 strains of H. influenzae or
M. catarrhalis isolated from middle ear effusions (Burman et al. 1994).
Significant rises in antibody titers were observed in only 2.7 and 0 % of healthy
children, respectively (Nohynek et al. 1995). Carriage of these bacteria may,
however, induce some positive results. In 121 children with acute respiratory
infection, positive antibody responses to H. influenzae or to M. catarrhalis

30
were found more often from children with simultaneous carriage of the bacterium
compared to those with negative pharyngeal sample (Nohynek et al. 1995).
For Chlamydia, a complement fixation assay (CF) and EIA can be used as a
screening test, and species-specific diagnosis is possible with microimmunofluorescence (MIF) (Ekman et al. 1993). Carriage of chlamydia has rarely been
reported to cause a serologic response in children, which suggests a relatively
high specificity of serology in chlamydia diagnostics (Normann et al. 1998).
However, the role of subacute or chronic infection as the cause of a seroresponse
is not yet well known (Tuuminen et al. 2000).
For mycoplasma, the serologic methods most commonly used are CF and
EIA. CF has been found to detect only 40 % of M. pneumoniae infections in
children < 4 years of age (Kleemola et al. 1993). Measurement of IgM antibodies
by EIA is considered more sensitive and is more specific, even in young children
(Waris et al. 1998).
5.4.1.4. Nucleic acid detection
A nucleic acid probe is a nucleic acid labeled with a compound that can be
visualized to detect a complementary sequence of DNA by hybridizing to it. In
this way microorganisms can be detected with probes hybridizing with the genes
of the organism. Too small a number of organisms is a limiting factor for the
routine use of gene probes and, therefore, PCR is increasingly used to amplify
the specific gene sequence. Thus, a very small number of pathogens can be
detected, which makes PCR not only sensitive but also prone to contamination
and false-positive results. On the other hand, natural inhibitors, e.g. porfphyrin
from heme in clinical samples, may inhibit the polymerase enzyme (Higuchi et al.
1989), resulting in false-negative results. Because PCR can detect fragments of
the DNA of infectious agents, it is assumed that previous administration of
antimicrobials does not influence the PCR results as much as it does the culture
results. However, no pneumococcal DNA was found in serum samples 48h
after initiation of intravenous ceftriaxone (Dagan et al. 1998). This is in
accordance with a Finnish study of pneumococcal bacteremia, in which
pneumolysin-PCR was negative in patients who had been on antimicrobials for
2-5 days before the sample was taken (Toikka et al. 1999a).
For detection of pneumococcal bacteremia and pneumonia, Ply-PCR from
whole blood, blood culture, serum, and buffy-coat samples has been used in
both adults (Rudolph et al. 1993, Salo et al. 1995, Lorente et al. 2000) and
children (Dagan et al. 1998, Toikka et al. 1999a). The sensitivity of the assay

31
has varied considerably. In children with invasive pneumococcal infection or
lobar or alveolar pneumonia, the pneumococcal genome was found in 15-43 %
of blood or serum samples by Ply-PCR (Dagan et al. 1998, Toikka et al. 1999a).
As compared with conventional serology and IC detection, blood-based PlyPCR was a more sensitive method. However, its clinical value is reduced by the
fact that, for optimal sensitivity, three blood fractions should be tested (Toikka
et al. 1999a). The specificity of the assay has been fairly good, though
oropharyngeal strains (Dagan et al. 1998) and -hemolytic streptococci, when
present in the sample (Kearns et al. 2000), may induce false-positive results. In
adults, another pneumococcal PCR, an assay to detect DNA of penicillin-binding
protein, has also been used for lung aspirates; the PCR had a higher sensitivity
than the standard culture (Ruiz-Gonzalez et al. 1997).
PCR assays for H. influenzae, M. pneumoniae, and C. pneumoniae have
been used in otitis media and pneumonia diagnostics for middle ear effusions
or NPA (Ueyama et al. 1995, Rayner et al. 1998, Jero et al. 1999, Waris et al.
1998). In pneumonia, however, the relevance of a positive PCR result from
the upper respiratory tract sample is questionable (Normann et al. 1998).
With regard to the diagnosis of acute infections in general, results based on
detection of very small amounts of certain organisms may need to be interpreted
with caution.

5.4.2. Viral agents

In contrast to frequent nasopharyngeal carriage of bacteria potentially causing
lower respiratory infections, carriage of viruses is less common. Therefore NPA
samples are more commonly used for viral diagnostics. Viral culture is the gold
standard (Bromberg and Hammerschlag 1987). More recently PCR assays for
several common respiratory viruses have also been developed (Paton et al. 1992),
though viral antigen detection from NPA samples is used more commonly
(Bromberg and Hammerschlag 1987). In clinical practice viral serology is seldom
needed, but in a research setting it may be useful. CF and EIA are used most
often, but the former has a relatively low sensitivity in childhood respiratory
infections (Korppi et al. 1986, Avila et al. 1990). For optimal diagnostics of
respiratory infections, the combined use of viral culture, direct antigen detection,
and determination of IgG antibodies in paired sera is considered necessary
(Korppi et al. 1993b).

32

5.4.3. Lung tap in childhood pneumonia

5.4.3.1. Earlier clinical experience
Lung tap, a method over a hundred years old (Leyden 1882) for obtaining a
representative sample, provides several advantages as compared with the methods
described above.
1) The sample is obtained straight from the infectious focus, the risk of
contamination with normal flora thus being avoided (Sinha and Huges
1966, Davidson et al. 1976).
2) Some organisms rarely cause bacteremic infections, but may be isolated
from the lung. In a study on Kenyan adults, H. influenzae was isolated
from 10 of 259 lung aspirates but from only 1 of 518 blood cultures
(Scott et al. 2000).
3) Culture of a lung aspirate has a very low false-positive rate; a positive
culture is considered strong evidence for bacterial infection (Shann 1986).
In Chile, a lung tap was performed on 13 infants with no clinical or
radiographic evidence of pneumonia; all 13 cultures remained negative
(Mimica et al. 1971).
According to data on more than 3500 children on whom lung tap was performed,
the method showed a relatively high positivity rate for bacterial agents (Shann
1986, Scott and Hall 1999, Vuori-Holopainen and Peltola 2001). A bacterial
etiology was revealed in 50 % of children with pneumonia overall (Vuori-Holopainen and Peltola 2001), the positive bacterial yield ranging from 10 to 96 %
(Vuori-Holopainen and Peltola 2001). Several reasons have been suggested for
false-negative results: the needle may have missed the consolidation, too small
a specimen or none may have been obtained, previous antimicrobial therapy
may have disturbed the growth, or the organism may not have grown in the
media used, or there has been a delay in culturing the specimens (Shann 1986,
Scott and Hall 1999). The positivity rate of the tap improves with the size of the
consolidation, dense peripheral consolidations being optimal (Zalacain et al.
1995). Other factors associated with higher sensitivity are the experience of the
person performing the tap, and lack of previous antimicrobial therapy (Zalacain
et al. 1995). In contrast to bacteria, few studies have explored viruses in lung
aspirates; therefore no conclusions can be drawn concerning the applicability of
lung tap in viral diagnostics (Vuori-Holopainen and Peltola 2001).
5.4.3.2. Risks of the procedure
Because lung tap is an invasive method, it is potentially harmful, the main risk
of the procedure being iatrogenic pneumothorax. In a review of 2 658 children
on whom lung tap was performed, 0.5 % required pleural drainage (Vuori-

33
Holopainen and Peltola 2001). The risk of clinically significant pneumothorax
may be reduced by puncturing the lung once only, by restricting the use of the
method to experienced operators, and by using a thin needle (Scott and Hall
1999). Other, even rarer, adverse events described in children are hemoptysis
(1.3 %) and subcutaneous emphysema (0.2 %) (Vuori-Holopainen and Peltola
2001). Pleuritic pain has rarely been reported. Three deaths associated with
lung tap have been described in children, in two of which the lung tap in itself
may have been the primary cause of death (Wesley et al. 1971, Cunanan et al.
1979, Shann et al. 1984). Notably, most of the lung-tap studies have included
only patients with consolidated pneumonia (Vuori-Holopainen and Peltola 2001).
Thus, in other types of pneumonia, the applicability and risks of the method are
difficult to assess.

5.5. Acute host response parameters

distinguishing viral from bacterial etiology
Erythrocyte sedimentation rate (ESR), peripheral white blood cell count (WBC),
and CRP are common host-response parameters used to differentiate bacterial
from viral infection. For ESR, a cut-off value 30 mm/H has been related to an
increased risk of bacteremia or radiologically verified pneumonia (McCarthy et
al. 1977). However, the response of ESR is rather slow (Triga et al. 1998) and
may be influenced by the size and shape of the erythrocytes, and the presence of
immunoglobulins (Gabay and Kushner 1999).
The WBC response is much more rapid than that of ESR. In children with
lobar or segmental pneumonia, the WBC count is at its highest during the first
two days of illness, and lowest on the fourth day (Triga et al. 1998). The most
common cut-off level used for differentiating between viral and bacterial etiology
is 15 x 109 /L (McCarthy et al. 1977, Putto et al. 1986). However, controversy
exists as to whether any level of WBC distinguishes a bacterial from a viral
respiratory infection reliably (Korppi et al. 1993c), because leukocytosis may
also occur in viral infection, especially if caused by adenovirus or Epstein-Barr
virus (Putto-Laurila et al. 1999). In a series of 154 febrile children, a WBC
count 15 x 109 /L was found in 67 % of cases with evidence for bacterial
etiology vs. in 13 % of those with probable viral disease (Putto et al. 1986).
CRP is an acute phase protein synthesized within 6-8 hours by the liver in
response to infection and a variety of inflammatory stimuli such as immunological,
traumatic, ischemic, and malignant tissue damage (Du Clos 2000). It is especially
useful for monitoring the presence of focal infections such as septic arthritis and
osteomyelitis, and their successful treatment (Unkila-Kallio et al. 1994, Kallio

34
et al. 1997). In childhood meningitis and bacteremic infections, serum CRP is a
sensitive and rapidly responding parameter differentiating bacterial from viral
etiology (Peltola 1982, Peltola and Rsnen 1982, Peltola and Jaakkola 1988,
Sormunen et al. 1999).
In respiratory infections, the ability of CRP to distinguish the etiology is less
evident, since CRP values of up to > 100 mg/L have been observed in infections
caused by adenovirus, influenza virus, or RS virus (Ruuskanen et al. 1985). In
some cases, however, undetected concomitant bacterial infection may have been
present.
Nevertheless, high CRP values are mostly associated with bacterial disease. If
a child has been ill for more than 8 to 12 hours, and the CRP value is < 40 mg/L,
a viral etiology is highly probable (Peltola 1983, Putto et al. 1986). Recently,
CRP was studied in children with bacteremic pneumococcal pneumonia. Initial
CRP values of 20 mg/L, 20 - 59 mg/L, 60 - 179 mg/L, and 180 mg/L were
observed in 15 %, 17 %, 42 %, and 26 % of patients, respectively (Toikka et al.
1999b). The CRP value increased for 24 hours in 74 % of patients during
antimicrobial treatment, but declined rapidly after the first day of therapy (Toikka
et al. 1999b).
More recently the use of other host response parameters such as procalcitonin
has been studied (Genrel and Bohuon 2000). In general, routine laboratory
tests have considerable, although limited, use in differentiating bacterial from
viral etiology, but they are of little value in specific identification of the causative
pathogen.

5.6. Antimicrobial therapy

The use of antimicrobials has steadily increased worldwide. Respiratory infections
account for 75 % of all community prescriptions, with approximately 818 million
prescriptions each year (Carbon and Bax 1998). Consequently, the antimicrobial
sensitivities of several bacteria have decreased markedly during recent years.
The spread of resistance reflects not only the appropriate use of antimicrobials
but also inappropriate prescribing. Antimicrobials are frequently prescribed for
non-bacterial infections, broad-spectrum regimens are overused, and dosages
that are too small or treatments that are too long are commonly prescribed
(Arason et al. 1996, Guillemot et al. 1998, Amsden 1999).

35

5.6.1. Treatment in relation to antimicrobial resistance

The in vitro resistance of bacteria is defined in terms of the minimal inhibitory
concentration (MIC), the lowest concentration that inhibits the growth of the
organism. For effective treatment, peak tissue levels of an antimicrobial should
be four to eight times the MIC values, and the concentration of the antimicrobial
should be higher than the MIC value for most of the time between doses (Shann
et al. 1987). However, standard susceptibility testing methods fail to take into
account the bactericidal activity of human serum and the potential for phagocytes
to enhance the antibacterial effects of antimicrobials intracellularly, both of which
may restrict the spread of infection. Therefore, in vitro resistance defined by
MIC values is not synonymous with failure of clinical treatment.
5.6.1.1. Streptococcus pneumoniae
Pneumococci are not intrinsically resistant to penicillin or to other commonly
used antimicrobials, but acquired resistance is increasing. The extent of the
resistance depends on the geographical area, the age of the patient population,
and the specimens investigated (Klugman 1990, Thornsberry et al. 1999, Forward
1999). S. pneumoniae isolates with penicillin MIC < 0.1 g/mL are defined as
penicillin-sensitive, strains with MIC 0.1-1.9 g/mL as intermediate resistant,
and strains with MIC 2 g/mL as highly resistant to penicillin (Friedland and
McCracken 1994). Approximately one-third of S. pneumoniae isolates in the
USA shows some resistance to penicillin. In Europe, the proportion varies from
1 to 50 % (Arason et al. 1996, Baquero 1995, Pradier et al. 1997), being highest
in Spain and lowest (1-3 %) in Scandinavia and in the United Kingdom (Pradier
et al. 1997). In Finland, the prevalence of highly resistant pneumococci is only
1 % (Pradier et al. 1997, Finres 1999).
There is little evidence that infections caused by penicillin-resistant
pneumococcus outside the central nervous system respond poorly to parenteral
penicillin or other -lactams (Friedland and McCracken 1994, Friedland 1995,
Pallares et al. 1995). In pneumonia, the concentration of penicillin in the lung is
equal to the concentration in the serum (Shann et al. 1987). With parenteral
penicillin, peak serum concentrations of up to 15-18 g/mL and concentration
1 g/mL for at least 18 hours are achieved (Shann et al. 1987). Hence, the
concentration achieved with parenteral penicillin is sufficient for treatment of
most pneumococcal infections.
A potential problem with the use of penicillin, however, is that it does not
effectively eradicate the carriage of penicillin-resistant pneumococci. Thus, at

36
least in theory, there is a risk of selection for carriage of these strains, and an
increase of invasive diseases due to these resistant strains (Klugman 1990).
This may be avoided by using parenteral therapy, with which higher antimicrobial
concentrations are obtained in nasopharyngeal mucosa (Klugman 1996).
Parallel with the spread of penicillin resistance, there has been an increase in
the resistance to other antimicrobials such as macrolides (Thornsberry et al.
1999, Forward 1999). In the USA, up to 23 - 31 % of pneumococcal isolates
are resistant to erythromycin (Thornsberry et al. 1999, Gay et al. 2000). In
Finland, the corresponding level is 7 % (Finres 1999), but, in the Helsinki area,
12 % (Dr M. Vaara, personal communication 2001). The clinical relevance of
high-level macrolide resistance suggests that potential treatment failures may
increase (Gay et al. 2000).
5.6.1.2. Influence of -lactamase production
Many H. influenzae and M. catarrhalis strains produce -lactamase, which
compromises the activity of penicillin, ampicillin, and amoxicillin, but does
not substantially affect the activities of cephalosporins (Zhanel et al. 2000).
The prevalence of -lactamase production has hardly changed in the last few
years (Thornsberry et al. 1999, Zhanel et al. 2000). In Canada and the United
Kingdom, up to 48.7 % and 15.1 % of H. influenzae and 94 % and 93 % of
M. catarrhalis respiratory tract isolates, respectively, produce -lactamase
(Felmingham et al. 1998, Doern et al. 1999, Zhanel et al. 2000). In the Helsinki
area, 20 % of all H. influenzae isolates and 93 % of the M. catarrhalis isolates
are -lactamase-positive (Dr M Vaara, personal communication 2001).
Although failures of amoxicillin treatment in otitis media caused by lactamase-producing H. influenzae and M. catarrhalis have been described
(Rosenfeld et al. 1994, Brook and Yocum 1995), -lactamase production has
not been shown to have as much influence on the management of pneumonia in
immunocompetent patients (Klugman 1996). In fact, in a Danish study, penicillin
and amoxicillin were effective in vivo in eradicating -lactamase-positive
M. catarrhalis from children with lower respiratory infections (Ejlertsen and
Skov 1996). Since pulmonary infections caused by M. catarrhalis are rare in
children, M. catarrhalis etiology need not to be considered in selection of
antimicrobials in cases of middle or lower respiratory infections affecting primarily
healthy children (Korppi et al. 1992a).

5.6.2. Antimicrobial treatment studies

A number of studies have compared two antimicrobial regimens in lower
respiratory infections. In adults, the efficacy of two -lactams, cefuroxime and

37
ampicillin (Pines et al. 1981), and a -lactam vs. a macrolide have been compared
(MacFarlane et al. 1983, MacFarlane et al. 1996). Cefuroxime led to a
satisfactory clinical response more often than ampicillin (Pines et al. 1981). In
contrast, a macrolide provided no advantage over standard amoxicillin
(MacFarlane et al. 1996).
Treatment of childhood pneumonia has been studied, especially in developing
countries. In those countries, co-trimoxazole, a treatment widely used in pediatric
pneumonia in the developing world, has been compared with procaine penicillin
and ampicillin (Campbell et al. 1988, Keeley et al. 1990, Sidal et al. 1994,
Straus et al. 1998). It was found effective in outpatient treatment. However, in
more severe pneumonia, procaine penicillin and ampicillin were more likely to
be effective. In probable pneumococcal pneumonia in Brazilian children, no
difference was found between a single dose of long-acting benzathine penicillin
and a 7-day course of procaine penicillin (Camargos et al. 1997).
From the developed countries, only limited data are available from randomized
studies. Atzithromycin was as effective as amoxicillin-clavulanate or erythromycin
for pediatric pneumonia (Roord et al. 1996, Harris et al. 1998). Intramuscular
penicillin and oral amoxicillin were found to be similar in effectiveness in the
early outpatient treatment of pediatric patients with presumed bacterial
pneumonia (Tsarouhas et al. 1998).

5.6.3. Length of antimicrobial treatment

The length of treatment has also been mostly empirical and rarely based on
comparative studies. Shorter courses of antimicrobials have only recently been
validated in various invasive and focal infections such as acute cystitis of women,
meningitis and osteomyelitis (MacFarlane et al. 1979, Lin et al. 1985, Peltola et
al. 1989, Norrby 1990, Peltola et al. 1997). In childhood upper respiratory
infections such as otitis media, sinusitis, and tonsillopharyngitis, short courses
are also effective (Pichichero and Cohen 1997, Cohen et al. 2000). In the latter,
6-day therapy with amoxicillin or 4-5-day therapy with various cephalosporins
or with azithromycin have been suggested as alternatives to traditional 10-day
penicillin therapy (Pichichero and Cohen 1997). A recently published metaanalysis of randomized, controlled trials for the treatment of otitis media suggests
that a 5-day course of short-acting antimicrobial is appropriate (Kozyrkyj et al.
1998). Apart from studies showing that a 3- or 5-day course of azithromycin is
as effective as a 10-day course of erythromycin or amoxicillin-clavulanate in
pediatric pneumonia (Roord et al. 1996, Harris et al. 1998), there have been
virtually no data on the length of treatment in childhood lower respiratory and
nonfocal infections.

38

5.6.4. Current guidelines for treatment of pneumonia

Because of the lack of randomized controlled trials, most treatment guidelines
are based on observations of the organisms in vitro susceptibility rather than on
proof of benefit from one antimicrobial over another (Jadavji et al. 1997).
However, in the absence of an etiologic diagnosis, the treatment of childhood
pneumonia is almost always empirical. -lactams have long been the first choice
for treating childhood pneumonia, but recently, in accordance with guidelines
for adulthood pneumonia (Am Thor Soc 1993), macrolides are often considered
the first choice for ambulatory patients (Ruuskanen and Mertsola 1999). No
clear consensus exists, since many other experts still favor amoxicillin as the
initial treatment (Klugman 1996). In Scandinavian countries, penicillin G is the
first choice in hospitalized children (Ruuskanen and Mertsola 1999). For patients
requiring intensive care, a combination of cefuroxime and a macrolide is often
recommended (Jadavdi et al. 1997).

6 AIMS OF THE STUDY

6.1. General aim
The aim of this study was to investigate the etiologic diagnostics and treatment
of common invasive bacterial infections of children, with special emphasis on
alveolar pneumonia.

6.2. Specific aims

1) To study the etiology of childhood community-acquired pneumonia and
other invasive suspected bacterial infections applying serologic methods.
2) To compare the clinical efficacies of parenteral penicillin and cefuroxime
for the treatment of common childhood infections requiring
hospitalization.
3) To compare the clinical efficacies of a 4-day treatment and a 7-day
treatment, using these -lactams.
4) To explore the advantages and disadvantages of lung tap in the etiologic
diagnostics of hospitalized childhood consolidated pneumonia.

39

7 PATIENTS AND METHODS

Table 5. Patients included in the study.

Paper I

170 children with suspected invasive bacterial infection

Paper II

154 children from paper I treated with randomized parenteral antimicrobials

(procaine penicillin N 75, cefuroxime N 79)

Paper III

154 children from paper I treated with parenteral antimicrobials,

the length of treatment randomized (4 days, N 73; 7 days, N 81)

Paper IV

One child with pneumonia caused by Moraxella osloensis

Paper V

34 children with community-acquired consolidated pneumonia

7.1. SE-TU study (I-III)

7.1.1. Study design
The SE-TU (Sepsis-tutkimus, Sepsis-research) study was an open,
randomized, prospective study of the etiology and treatment of common
childhood infections, in which two antimicrobial regimens and two lengths of
treatment were compared. The study was carried out during two time periods
(February 1988-October 1989, and March 1991-October 1993) at the Childrens
Hospital, University of Helsinki, and Aurora Hospital, Helsinki. The Ethics
Committees of both participating hospitals approved the SE-TU study protocol,
and oral consent was obtained from the guardian of each patient. From a
computer-based list, one of the following four treatments was chosen: 1) procaine
penicillin 50,000 IU/kg/day i.m. for 4 days, 2) procaine penicillin 50,000 IU/kg/
day i.m. for 7 days, 3) cefuroxime 100 mg/kg/day i.v. divided into 3 doses for 4
days, or 4) cefuroxime 100 mg/kg/day i.v. divided into 3 doses for 7 days. If the
child was known to be allergic to the randomized drug, the other alternative
was chosen. If clinical recovery was rapid, the 7-day treatments were completed
at home with oral penicillin V or cefuroxime axetil, respectively.

40
If the child showed symptoms and signs suggesting an invasive bacterial
infection (McCarthy et al. 1982), he or she was included regardless of the
results of laboratory or radiographic investigations. The exclusion criteria were:
age < 3 months, meningitis, osteoarticular, or urinary tract infections, patients
in the oncology, trauma, surgery, and intensive care units. CRP was measured
on admission (Peltola et al. 1984). If the value was 80 mg/L, the randomized
antimicrobial was instituted; if it was 20-79 mg/L, the measurement was repeated
within 8-12 hours, and the child was included if there was an increase of at least
50 %. If CRP remained < 20 mg/L, antimicrobial was begun only if the patient
deteriorated. Blood cultures, NPA samples, and acute- and convalescent-phase
serum samples were collected. The etiology of infections was studied with an
extensive panel of antigen and antibody assays detecting seven bacteria, 15 viruses,
and one protozoan. The patients included in the SE-TU study and randomized for
parenteral antimicrobials are presented in the study profile in Figure 1.
Originally, 178 patients fulfilled the inclusion criteria. The final number of
patients in the etiologic analysis (Paper I) was 170. Ninety-seven children (57 %)
were boys and 73 (43 %) were girls. Their ages ranged from 3 months to 15
years, the mean age was 3.8 years (Figure 2).
The treatment analyses (Papers II and III) comprised 154 children. The reasons
for exclusion of eight children enrolled in the etiologic analysis (Paper I) and of
24 patients in treatment analyses (Papers II, III) are described in Figure 1.
The SE-TU Study Profile
Patients enrolled
N 178
Patients with underlying
disease excluded
N8

Patients with randomized

treatment
N 170

Penicillin N 85
4-day N 42
7-day N 43

Cefuroxime N 85
4-day N 39
7-day N 46
6 patients
excluded**

10 patients
excluded*

Study completed
Pen 4-day
N 36

Study completed
Pen 7-day
N 39

Study completed
Cfm 4-day
N 37

Study completed
Cfm 4-day
N 42

* Four cases of likely viral disease, four prolonged treatment for otitis media or streptococcal tonsillitis, one case of probable listeriosis,
one with change of treatment for undetermined reasons.
** One case of likely viral disease, two patients refused to stay in hospital, and treatment was continued with another antimicrobial,
two cases of prolonged treatment for otitis media or streptococcal tonsillitis, once insurmountable problems with i.v. access.

Figure 1. Study profile of the SE-TU studies (I-III).

41

Figure 2. Age distribution of 170 children in the SE-TU series (I).

7.2. KE-TU study (IV, V)

7.2.1. Study design
The KE-TU (Keuhkokuume-tutkimus, Pneumonia-research) study was
another prospective study on the use of lung tap in pediatric pneumonia carried
out at the Helsinki University Central Hospital, Hospital for Children and
Adolescents from December 1997 to December 2000. The Ethics Committee
of the hospital approved the study, and written consent was required from the
parents. The diagnosis of pneumonia was based on simultaneous findings of at
least a history of fever and/or respiratory symptoms and consolidated infiltrates
compatible with alveolar pneumonia in the chest radiograph. In all, 47 patients
were asked to participate, but, since 13 refused, the study included 34 hospitalized
children, in all of whom a lung tap was performed. Postoperative, oncologic,
and immunocompromised patients, and those with a bleeding diathesis or in
intensive care were excluded, as were those < 3 months of age. In paper IV, a
6-year-old girl with bilateral pneumonia caused by Moraxella osloensis is
described. Paper V comprises 34 children with consolidated pneumonia. The
mean age was 5.1 years, their ages ranging from 10 months to 14.0 years. Twenty
(58 %) of the patients were boys and 14 (42 %) were girls.

42

7.2.2. Technique of lung tap

The consolidated lesion was localized in two planes according to chest
radiographs and physical signs. The lung tap was performed in the outpatient
department, at the bedside without fluoroscopy or computed tomography, and
before antimicrobial therapy was instituted. The skin was anesthetized with
lidocaine (Emla) ointment. During the procedure, the child was held tightly in
a sitting or supine position. After sterilization of the skin, a standard thin needle
(22G) was attached to a 5-mL syringe containing 2.5 mL of saline, and inserted
supracostally into the consolidated lung. To increase the volume of the aspirate,
1.5 mL of saline was injected into the lung, and the needle was removed
immediately during continuous suction. The whole procedure lasted < 10
seconds. After the lung tap, the patient was monitored closely and the respiratory
rate was checked repeatedly. A control chest radiograph was taken within 2
hours after the procedure (Perlmutt et al. 1986).

7.3. Specimens and microbiologic methods

7.3.1. Conventional microbiology (I-V)
Blood cultures were obtained from all patients by peripheral venipuncture. Eleven
percent of the patients in the SE-TU series and 15 % of patients in the KE-TU
study had received antimicrobials prior to hospitalization. For culture, the routine
(BacT/Alert) aerobic blood culture medium was used. The oropharyngeal
samples obtained on admission were cultured routinely. In the SE-TU series (IIII), these samples were available only from patients with tonsillitis, but, in the
KE-TU series (IV, V), from all patients. For viral antigen detection indirect
immunofluorescence from NPA samples was performed, using seven monoclonals
(Respiratory Panel 1, Light Diagnostics, Temecula, CA, USA) against the
following viruses: RS, adeno, parainfluenza 1-3, and influenza A and B (Ukkonen and Julkunen 1987). NPA samples were collected from 122/170 patients of
the SE-TU series (I) and from 32/34 patients of the KE-TU series (V).

7.3.2. Serology (I-III)

For serology, acute-phase serum samples were collected on admission to hospital
and the convalescent sera 2-3 weeks later. All samples were stored at 70C
until assayed. Microbiologic diagnoses were based on a single high value (IgM),
a rise in the specific antibody titer in paired sera, or demonstration of specific
ICs in acute or convalescent serum (Table 6). The antibody assays for S.

43
pneumoniae, H. influenzae, and M. catarrhalis used in this study have been
validated in healthy children and in young adults with common colds; positive
results were found 2.7 % of cases (Korppi et al. 1989, Nohynek et al. 1995,
Korppi and Leinonen 1998, Mkel et al. 1998). Samples for determination of

Table 6. Serologic methods used (I-III).

Agent

Antigen used

Assay

Diagnostic criteria

Reference

S. pneumoniae

Ply

EIA

2-fold increase

Jalonen et al. 1989

ICs

Ply

EIA

GM+2SD* (550)

Leinonen et al.1990,Korppi and

Leinonen 1998

ICs

C-PS

EIA

GM+2SD (400)

Leinonen et al.1990,Korppi and

Leinonen 1998

H. influenzae,
nontypable

whole cell

EIA

3-fold increase

Burman et al.1994

M. catarrhalis

whole cell

EIA

3-fold increase

Burman et al.1994

Chlamydiae

common group
antigen

CF

4-fold increase

Ukkonen et al. 1984

C. pneumoniae

type specific
elementary bodies

micro-IF

4-fold increase/
presence of IgM

Ekman et al. 1993

C. trachomatis

type specific
elementary bodies

micro-IF

4-fold increase/
presence of IgM

Ekman et al. 1993

whole cell

CF
EIA

4-fold increase
3-fold increase

Ukkonen et al. 1984

Kleemola et al. 1990

T5, T8 CPS

EIA

Fattom et al.1990,1993

alphatoxin

EIA

2-fold increase,
GM+2SD (25)
GM+2SD (500)

Respiratory and
other viruses**

CF

4-fold increase

Ukkonen et al. 1984

Epstein-Barr virus

IF
EIA

presence of IgM
IgG avidity <25%

Hedman et al.1993
Hedman et al.1993

Human herpes virus 6

IF

4-fold increase

Linnavuori et al. 1992

Toxoplasma gondii

CF

4-fold increase

Ukkonen et al. 1984

EIA

IgG avidity <15%

Hedman et al. 1993

M. pneumoniae

S. aureus
ICs

Julander et al.1983

* GM+2SD, geometric mean + 2 standard deviations

** Viruses included in the CF panel: influenzae A and B, parainfluenzae 1, 2, and 3, adenovirus, RSV, coxsackie B5,
herpes simplex, rotavirus, cytomegalovirus, varicella zoster and mumps virus

44
S. pneumoniae, H. influenzae, M. catarrhalis and S. aureus antibodies and
pneumococcal and staphylococcal ICs were available from all (N 170) patients,
and for chlamydia, mycoplasma, EBV, and HHV-6 serology from 169, 127,
165, and 161 patients, respectively. A CF assay for viruses and detection of
Toxoplasma gondii antibodies was possible from 105 patients.

7.3.3. Lung aspirates (IV, V)

The presence of leukocytes in the stained specimen was recorded; any leukocytes
seen in the lung aspirate samples at 100x magnification were considered indicative
of a representative sample (Zalacain et al. 1995). Besides conventional staining
and culture methods, PCR techniques and commercial RNA-DNA hybridization
assays were also used to detect bacteria. The specific bacteria searched for with
PCR were S. pneumoniae, H. influenzae, C. pneumoniae and M. pneumoniae
(Table 7). The Gram-negative bacillus isolated from a cultured needle (paper
IV) was further identified by DNA sequencing (Jalava et al. 1995, Jalava and
Eerola 1999).
Virus detection from lung aspirates was performed by means of viral culture,
antigen detection (Respiratory Panel 1, Light Diagnostics, Temecula, CA, USA)
and PCR detecting RNA specific for entero- and rhinoviruses (Lnnrot et al.
1999). A summary of the microbiologic methods used and the number children
on which each test was performed are presented in Table 7. If the amount of the
lung aspirate sample was insufficient, priority was given to standard microbiologic
studies and aspirate was frozen (-70C) for PCR studies only after taking samples
for the staining and culture.

7.3.4. Serotyping and susceptibility testing of

Streptococcus pneumoniae (V)
The antimicrobial sensitivity of the S. pneumoniae strains was measured by the
standard disk diffusion method. If the test indicated lowered antimicrobial
susceptibility, MIC-concentrations were determined by the E test (Forward 1999).
Pneumococcal isolates were serotyped by counter-immuno-electrophoresis and
by the latex agglutination test for neutral polysaccharides of types 7 and 14,
utilizing omniserum, serum pools, and type-, group-, and factor-specific antisera
(Statens Serum Institut, Copenhagen, Denmark). In unclear cases, the capsular
quellung reaction was used for confirmation of the result.

45
Table 7. Microbiologic methods used for lung aspirates (IV, V).
Pathogen

Method

No of tests
perfromed

Reference

Bacteria

Staining (acridine orange/gram)

Culture (Bact/Alert)
Needle culture

34/34
34/34
33/34

Baron et al.(ed.) 1994,

Lauer et al.1981

S. pneumoniae

Pneumolysin PCR

32/34

Kontiokari et al. 2000

DNA-RNA hybridization
(Accuprobe)

29/34

C. pneumoniae

PCR

32/34

Described in paper V

H. influenzae

PCR

32/34

Hobson et al. 1994

DNA-RNA hybridization
(Accuprobe)

27/34

M. pneumoniae

PCR

28/34

van Kuppeveld et al. 1992

Vesanen et al. 1996

M. osloensis

DNA sequencing

1/34

Jalava et al.1994, 1995

Viruses

Culture

33/34

Adenovirus
Influenza A and B
Parainfluenzae 1-3
RSV

IF (Light Diagnostics)
IF (Light Diagnostics)
IF (Light Diagnostics)
IF (Light Diagnostics)

26/34
26/34
26/34
26/34

Enterovirus
Rhinovirus

PCR
PCR

28/34
28/34

Lnnrot et al. 1999

Lnnrot et al. 1999

7.4. Criteria for etiology (I-V)

Papers I-III. An infection was considered to be of bacterial origin if at least one
of the following criteria was fulfilled: positive blood culture, a significant antibody
response or specific ICs present in sera samples (Table 6), or Streptococcus
pyogenes isolated from the pharyngeal sample. The diagnosis of viral infection
was based on virus antigen detection from NPA or a significant antibody response
(Table 6). A mixed viral-bacterial infection was diagnosed when there was
microbiologic evidence for bacterial and viral infection in the same patient. Dual

46
and triple infections were considered to be present when evidence for two or
three bacteria or viruses was found in the same patient. The percentages were
counted from all patients, although some assays could not be performed from
the whole series.
Papers IV and V. The etiology of pneumonia was considered definitive
when a pathogen was detected in a normally sterile sample (blood, lung aspirate,
or puncture needle). The etiology was considered presumptive if a viral antigen
was detected in the NPA, or if S. pyogenes was isolated from the pharyngeal
sample of a patient with tonsillitis.

7.5. Radiologic assessment (I-V)

Papers I-III. Chest radiographs were obtained on admission and, if the finding
suggested pneumonia, a control radiograph was taken five days later. The
radiographs were reviewed by a pediatric radiologist unaware of the results of
microbiologic assays or clinical signs. Patients with alveolar consolidation, either
lobar or bronchopneumonia, were included in the pneumonia group, whereas
those with respiratory symptoms but no clear alveolar consolidation, such as
diffuse and increased interstitial infiltrates, were included in the group of other
respiratory infections.
Papers IV and V. The diagnostic criteria for pneumonia were a consolidated
alveolar infiltration compatible with pneumonia observed on admission, together
with fever and/or respiratory symptoms. Radiologically pneumonia was defined
as a consolidated infiltrate, which diminished or disappeared during the followup. Chest radiographs were interpreted by the radiologist on duty, and reevaluated by another pediatric radiologist unaware of the etiologic diagnosis
and previous radiologic results. Check-up radiographs were similarly interpreted.

7.6. Statistical methods (II, III, V)

In the treatment analyses, the primary endpoint for successful treatment was
uneventful recovery with no change in the randomized antimicrobial (II) or in
the length of treatment (III). A sample size analysis showed that a total of 142
patients would have disclosed a possible 20 % difference in the effectiveness of
the two treatments at a level of p = 0.05 (two-sided test) with an 80 % power
(II, III). For categorical comparisons, the chi-squared contingency analysis and
Fishers exact test were used. For continuous variables, analysis of variance and
Students t tests were used to compare the mean values (II, III, V). All tests
were two-sided. P < 0.05 was considered statistically significant.

47

8 RESULTS
8.1. Etiology in the four clinical manifestations (I)
The patients in this analysis were divided into the following four categories:
1) Pneumonia (N 79). Children with acute respiratory symptoms and signs
and alveolar consolidation in a chest radiograph.
2) Other respiratory infections (N 30). Patients with respiratory symptoms
and signs, but without a clear radiologic finding suggesting alveolar
pneumonia.
3) Sepsis-like infections (N 37). Children with high fever ( 38.5C), and an
initial CRP value 100 mg/L but no respiratory symptoms or evidence
for a focal infection.
4) Other acute infections (N 24). Cases with no respiratory symptoms, high
fever, or clearly increased CRP ( 100 mg/L) value but in which the
clinical status warranted antimicrobials (e.g. cellulitis, lymphadenitis,
sepsis-like disease with either lower fever or CRP < 100 mg/L).
Evidence for a microbial etiology was found in 61 %, 73 %, 68 %, and 42 % of
the groups of pneumonia, other respiratory, sepsis-like, and other acute infections,
respectively (Table 8). Bacteria alone were found in 49 %, 50 %, 46 %, and 38 %
of children with pneumonia, other respiratory, sepsis-like, and other acute
infections, respectively, while mixed bacterial-viral infections were found in 11 %,
7 %, 3 %, and 0 % of cases, respectively. In all groups combined, bacterial etiology
alone, or concomitantly with viruses was found in 92 children. The overall
distribution of bacterial, mixed, and viral etiologies in the various clinical
manifestations in patients with etiology identified is shown in Figure 3.
S. pneumoniae was the most common bacterial pathogen found (N 62), being
present in 46 %, 30 %, 24 % , and 33 % of pneumonia, other respiratory
infections, sepsis-like infections, and other acute infections, respectively. Either
alone or combined with other agents, it accounted for 75 %, 41 %, 36 %, and
80 % of cases with an identified etiology. The next commonest bacterial pathogens
identified were nontypable H. influenzae and C. trachomatis (Table 8). Most
bacterial diseases were diagnosed by serology alone, since blood culture was
positive in only 5 %.

48
Table 8. Etiologic agents identified in various clinical manifestations (I).
Disease

Etiology identified
N %

Agent

N
%
(in each category)

Pneumonia
(N 79)

48

61

S. pneumoniae alone
+H. influenzae
+RSV
+Other(s)
H. influenzae alone
+HHV-6
+RSV
M. catarrhalis alone
+Parainfluenza 3
C. trachomatis
M. pneumoniae
None

24
2
2
8
2
1
1
1
1
5
1
31

30
3
3
10
3
1
1
1
1
6
1
39

Other respiratory
infection
(N 30)

22

73

S. pneumoniae alone
+M. catarrhalis
+Adenovirus
+RSV
+Other(s)
S. aureus alone
S. pyogenes
H. influenzae type b
+C. trachomatis
Adenovirus
Other(s)
None

4
1
1
1
2
3
3
1
1
3
2
8

13
3
3
3
7
10
10
3
3
10
7
27

Sepsis-like infection
(N 37)

25

68

S. pneumoniae alone
+Other(s)
H. influenzae
M. catarrhalis
Adenovirus
Other(s)
None

7
2
4
2
4
6
12

19
5
11
5
11
16
32

Other acute infection

(N 24)

10

42

S. pneumoniae alone
+Other(s)
S. aureus
Influenza b
None

6
2
1
1
14

25
8
4
4
58

49

Figure 3. The etiologic spectrum in various clinical manifestations among the cases
with an identified etiology (N 105) (I).

8.2. Patients with randomized treatment (II, III)

8.2.1. Characteristics and bacterial etiology (II, III)
Initial characteristics of the 154 evaluable patients are presented in Table 9.
Most of the patients had high fever, together with significantly increased CRP,
WBC, and ESR values. On admission, in 80 % patients, CRP was 80 mg/L,
while in 9 % of the cases CRP increased by 50 % in the following 6-8 hours.
Microbiologic evidence for a bacterial etiology was obtained in altogether 55 %
of the cases. (Table 10). Serologic evidence for S. pneumoniae infection was
found in 59 cases and for Chlamydia or Mycoplasma infection in 13 cases.
On admission, the patients in the penicillin group tended more often to have
cough and diarrhea than the children in the cefuroxime group. Vomiting and
pneumonia was found more often in the penicillin group and sepsis-like disease
in the cefuroxime group. Otherwise, the treatment groups were almost identical
(Table 9).
The symptoms and signs of the children in the 4-day and 7-day groups were
similar on admission, although mixed bacterial-viral infections were more
common in the children in the 4-day group (14 % vs. 2 %). In contrast, single
bacterial infections were more common in the 7-day group (47 % vs. 27 %).

50
Table 9. Initial characteristics and disease manifestations in the 154 evaluable
patients in the SE-TU studies (II, III).

Girls
Boys
Age (yrs)

Study II (Pen vs. Cfm)

Study III (4- vs. 7-day)

Pen (N 75)
Cfm (N 79)
Mean/ % Range Mean/ % Range

4-day (N 73)
7-day (N 81)
Mean/ % Range Mean/ % Range

43
57
3.9

0.7-11.8

46
54
3.9 0.3-14.0

Clinical signs / symptoms

Body temperature (C) 39.2 36.2-40.8
Cough (%)
61
Vomiting (%)
34*
Diarrhea (%)
9

39.1 36.7-41
39.1
44
18*
5

Pretreatment
antimicrobials (%)

Laboratory parameters
WBC (x109/L)
21.7
ESR (mm/H)
53
CRP (mg/L)
137
CRP 80 mg/L (%)
83
CRP 50% increase
7
in 8-12 hours (%)**
Disease manifestation
Pneumonia
Other respiratory
infection
Sepsis-like infection
Other acute infection

10

3.6-42.5
8-123
23-400

22.3 8.3-45.2
55
2-131
127 12-334
77
11

49
51
3.8

0.7-14

39.1 36.2-40.9
51
25
7
11

21.6 3.6-45.2
59
2-131
131 33-270
82
8

40
60
3.9 0.3-12.7

39.2
54
25
8

All patients
Mean/%

44
56
1.4

36.7-41

39.1

22.4 9.0-44.4
50
5-115
132 12-334
78
10

22
55
132
80
9

55*

39*

46

47

47

15

18

15

17

16

17*
13

28*
15

25
14

21
15

23
14

* p < 0.05
** Initial CRP value < 80 mg/L.

51
Table 10. Bacterial etiology of the 154 patients in the SE-TU studies (II, III).

Study II (Pen vs. Cfm)

Study III (4- vs. 7-day)

Pen (N 75)
%

Cfm (N 79)
%

4-day (N 73)
%

7-day (N 81)
%

Total
%

Bacteria in all
single
dual / triple
mixed with virus(es)

51
39
4*
8

58
37
14*
88

49
27*
8
14*

59
47*
10
2*

55
38
9
8

Etiology disclosed in all

56

68

62

63

62

Specific agents
N
S. pneumoniae alone
20
with virus
3
with Chlamydia / Mycoplasma 1
with H. influenzae /
2
M. catarrhalis
with other bacteria
0

N
19
6
4
2

N
15
7
2
1

N
24
2
3
3

Total
39
9
5
4

H. influenzae
with virus
with Chlamydia

2
2
0

5**
0
1111

1***
2
0

6***
0
1

7
2

M. catarrhalis alone
with virus
with Chlamydia

2
1
0

1
0
1

0
1
1

3
0
0

3
1
1

Chlamydia / Mycoplasma

Other bacteria

* p < 0.05
** 2 type b
*** 1 type b

52

8.2.2. Antimicrobial therapy, length of treatment, and

recovery (II, III)
Seventy-five patients were treated with penicillin, and 79 with cefuroxime (Table
11). Clinical recovery and duration of fever or the need for antipyretics in the two
groups were similar (Table 11). Acute host response parameters, such as CRP,
WBC, and ESR, normalized at equal rates during treatment (Figure 4). No
difference was found in potentially drug-related adverse effects, including diarrhea,
nausea, and eczema (Figure 5). Most children suffering from nausea in the penicillin
group had this symptom already on admission, before starting the medication
(data not shown). In two patients initially treated with penicillin, the scheduled
antimicrobial regimen was changed during treatment, and one patient in each
treatment group was rehospitalized within 1-2 days, and a different antimicrobial
was instituted (Table 11).
The randomized length of treatment was 4 days in 73 cases, and 7 days in 81
cases. Medication was completed parenterally as scheduled in 97 % of the patients
in the 4-day group, and in 62 % of those in the 7-day group. The remaining
Table 11. Realization of the randomized treatments, recovery, and outcome of
patients in the SE-TU studies (II, III).
Pen 4
N 36

Pen 7
N 39

Cfm 4
N 37

34
0

28
11

37
0

22
20

Fever (> 37.5C) / required antipyretics

day 2
17 (47%)
day 4
4 (11%)

15 (38%)
6 (15%)

12 (32%)
3 (8%)

15 (36%)
6 (14%)

141
50

163
57

141
53

141
46

Treatment completed but

rehospitalization with another
antimicrobial in 1-2 days

Patient rehospitalized within 1 month

Visited outpatient clinic /

private physician in 1 month

Realization Treatment completed parenterally

Treatment completed orally
Treatment changed during
treatment
Recovery

CRP, mean (mg/L)

day 2
day 4
Outcome

Cfm 7
N 42

53
patients in the latter group completed their medication orally at home (Table 11).
Recovery during treatment was similar in the two groups, with no difference in
symptoms or signs, or rate of normalization of laboratory parameters (data not
shown). In two patients in the 4-day group, the randomized length of treatment
was prolonged with another regimen, while one patient in each group was
rehospitalized within 1-2 days, and antimicrobial was reinstituted (Table 11).
The results were similar, when the data was looked between the four subgroups
with different regimens and different lengths of treatment used (Table 11).

Figure 4. WBC, CRP, and ESR of patients in the Pen vs. Cfm study (II). Values are
presented as means with standard errors, p = NS.

54

Figure 5. Symptoms and signs during recovery in the Pen vs. Cfm study, p = NS (II).

8.2.3. Clinical outcome (II, III)

Realization of the randomized treatments and outcome of the patients are
presented in Table 11. In two cases, the randomized antimicrobial was changed
during treatment and the course of therapy was prolonged. The first case was a
2-year-old girl with periorbital cellulitis randomized to the 4-day penicillin group.
Because of her slow response to procaine penicillin, the antimicrobial was
changed on the third day to cefuroxime for an additional four days. The etiology
remained unknown.
The other case was a 5-year-old girl with pneumonia, also randomized to
the 4-day penicillin group. During hospitalization, treatment was changed to
erythromycin. Blood culture was negative, but C. trachomatis infection was
diagnosed by serology. In both cases, penicillin treatment was considered
ineffective.
Two children completed their treatment as scheduled, but were rehospitalized
within 1-2 days. A 4-year-old girl with bilateral pneumonia was randomized to
the 4-day cefuroxime group. The initial response was good but two days after
the end of therapy, fever and respiratory symptoms reappeared. The child was
readmitted and treated with parenteral penicillin. S. pneumoniae and HHV-6
were identified by serology.
The other case was a 2-year old boy with bacteremic pneumococcal pneumonia
(penicillin MIC < 0.1 g/mL). After procaine penicillin for seven days, fever

55
and respiratory symptoms reappeared. Blood culture was negative and cefuroxime
was administered for five days. Both viral antigen detection from NPA obtained
on first admission and viral serology from paired sera were negative. The symptoms
may have been caused by an undetected secondary infection.
Neither the antimicrobial used nor the length of therapy influenced the
frequency of visits to the outpatient clinic or some other physician within one
month after hospitalization (Table 11). All the patients recovered.

8.3. Lung tap (IV, V)

8.3.1. Sample validity and overall yield
Lung tap was performed on 34 children, in 26 of whom an accurate sample with
leukocytes was obtained. In most cases, the volume of the lung aspirate ranged
from a few drops to a few milliliters. The lung aspirate disclosed the etiology in
59 % of the cases. There were 16 single bacterial infections, two dual bacterial
infections, one mixed bacterial-viral infection, and one viral infection. Among
the 26 patients with a representative sample, the etiology was established in
69 %. Patients with a positive lung aspirate were significantly younger (mean 3.6
vs. 7.0 years, p = 0.02), and had a higher CRP (216 mg/L vs. 134 mg/L, p= 0.01)
and ESR (87 mm/H vs. 63 mm/H, p = 0.01) on admission than those with a
negative sample. However, there were no differences either in the WBC (20.6 x
109/L vs. 17.1 x 109/L) on admission, or in the duration of symptoms, or degree
of fever on admission (data not shown).

8.3.2. Etiology of consolidated pneumonia

S. pneumoniae was detected in the lung aspirate in 53 % of the cases, in 15
cases as a single pathogen, once with enterovirus, once with C. pneumoniae,
and once with Moraxella osloensis. M. pneumoniae pneumonia and viral
pneumonia (RSV or parainfluenza) were each detected once (Table 12). Blood
culture was positive for S. pneumoniae only twice (Table 12).
RSV was detected from the nasopharynx in three children, with simultaneous
isolation of S. pneumoniae by lung tap. In these cases, no viruses were found
from the lung tissue. In one case, conventional methods suggested S. pyogenes
tonsillitis without concomitant finding from lung aspirate. S. pneumoniae was
twice identified simultaneously from blood and lung.

56
Combining all the above-mentioned diagnoses, lung tap increased the proportion
of patients with evidence of etiology from 15 % (5/34) to 62 % (21/34). The
proportion of cases in which a definitive etiology was established increased from
6 % (2/34) to 59 % (20/34).
Table 13 shows the age, CRP, WBC, and body temperature of the patients
with a positive lung aspirate. All the parameters varied widely, regardless of the
etiology identified. The radiographic findings caused by the different agents
identified by lung tap are presented in Figure 6. Radiographs are presented from
the cases in which different etiologies were identified, but, for pneumococcal
pneumonia, only one typical chest radiograph was chosen (Figure 6A).
8.3.2.1. Moraxella osloensis (IV)
An atypical pathogen, M. osloensis, was isolated by lung tap from a 6-year-old
girl with a 10-day history of cough and a 4-day history of abdominal pain. On

Table 12. Positive results obtained by different methods in the 20 cases in which
the etiology was detected.
Lung
aspirate
staining*

Lung
Blood Pnc** Pnc-RNA Hi**
aspirate culture PCR hybridiPCR
culture
zation

leuk+++
leuk+++
leuk+++
leuk+++
leuk+++
leuk++****
leuk++****
leuk++****
leuk++
leuk++
leuk++
leuk++
leuk++
leuk++
leuk+****
leuk+
leuk+
leuk+
-

Pnc
Pnc
Pnc
Pnc
Pnc
Pnc
Pnc
Pnc
Pnc
-

*
**
***
****

M. osloensis Pnc
Pnc
Pnc
-

+
+
+
ND
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
ND
+
+
+
+
+
+
+
+
ND
-

ND
-

Hi-RNA C.pn**
hybridi- PCR
zation
ND
ND
ND
ND
-

ND
+
-

M.pn**
PCR

ND
ND
ND
+
-

Rhino
PCR

Entero
PCR

Viral
culture

ND
ND
ND
-

ND
+
ND
ND
-

+***
ND
-

-, no leukocytes; +, 1-2 leukocytes; ++, 3-5 leukocytes; +++, >5 leukocytes seen with 100x magnification.
Pnc, S. pneumoniae; Hi, H. influenzae; C.pn, C. pneumoniae; M.pn, M. pneumoniae.
Parainfluenza or RS virus, final typing impossible.
Gram+cocci in staining

57
Table 13. Age, C-reactive protein (CRP), white blood cell count (WBC) , and
body temperature on admission in patients in whom the etiology was identified
by lung tap (IV, V).
Patient

Age
years

CRP
mg/L

WBC
109/L

T
C

Pathogen found

Method

4
5
7
8
9
11
12
13
14
15
17
19
20
21
22
23
24
27
28
29

2.9
0.8
8.7
12
1.1
1.6
3
6
1.5
1.8
1.3
14.8
1
2.5
1.8
3.2
2.1
4
6.1
1.3

279
264
234
220
102
314
195
16
131
345
186
222
290
330
144
204
174
196
260
232

23.2
28.6
25.8
17.7
17.2
8.2
33.1
27.9
24.3
22.9
20.9
6.0
25.7
14.3
17.7
13.5
19.4
15.5
21.8
29.4

39.5
40.5
39
39.7
38
40
37.3
38.9
39.2
37.3
38.3
39.4
40
38
36.6
38
39.7
38.2
37.6
38.2

Pnc
Pnc
RSV/parainfluenza
Pnc
Pnc
Pnc
Pnc, C. pneumoniae
Pnc
Pnc
Pnc
Pnc
M. pneumoniae
Pnc
Pnc, enterovirus
Pnc
Pnc
Pnc
Pnc
M. osolensis, Pnc
Pnc

culture
culture
culture
culture
PCR
culture
PCR
PCR
culture
culture
culture
PCR
PCR
culture, PCR
PCR
culture
PCR
PCR
culture, PCR
culture

Mean

6.8

217

20.7

38.7

6-33.1

36.6-40.5

Range

0.8-14.8

16-345

admission, she had fever (37.6 C, a few hours later 39.7 C), respiratory rate
48 /min, and oxygen saturation 95 %. The CRP value was 260 mg/L, WBC
21.8 x 109/L, and ESR 127 mm/H. A chest radiograph revealed bilateral
consolidation in the lower lobes (Figure 6B). Urine analysis, throat culture,
viral antigen detection from NPA, and blood culture were negative, while
culturing of the needle yielded a pure culture of penicillin-sensitive M. osloensis.
A likely concomitant pneumococcal infection was diagnosed afterwards by PCR,
pneumococcal DNA-RNA hybridization still remaining negative.

8.3.3. Adverse events

In the check-up radiograph, pneumothorax was detected in six patients; five
were considered iatrogenic, of whom one had concomitant pneumomediastinum.
All these cases resolved spontaneously without any treatment or pleural drainage.
The hospital stay and the length of antimicrobial therapy were not prolonged.
Neither the length of the history, the pretap symptoms nor the signs or values of

58

2.9-year-old boy; fever for 1 day. S. pneumoniae

6.1- year-old girl; cough for 10 days, abdominal pain for 4 days.
M. osloensis+ S. pneumoniae

Figure 6. Chest radiographs of patients with etiology disclosed by lung tap. An example
of typical pneumococcal pneumonia (A) and all the other cases, in which different
etiologies were identified, are presented (B-F).

59

3.0- year-old boy; fever and vomiting for 4 days.

S. pneumoniae + C. pneumoniae

2.5- year-old girl; fever, cough, vomiting for 2 days.

S. pneumoniae + enterovirus

Figure 6. Continued.

60

14.8- year-old boy; fever and cough for 5 days. M. pneumoniae.

8.7- year-old girl; fever, vomiting, headache, and pain on right side of
thorax for 2 days. RSV or parainfluenza virus.

Figure 6. Continued.

61
acute-phase parameters on admission distinguished the patients with and without
pneumothorax (data not shown). Nor did the frequency of obtaining a valid
sample correlate with that of post-tap pneumothorax (data not shown).

8.4. Diagnostics of pneumococcal pneumonia

with different methods (I, V)
Study I comprised 79 children with evidence of alveolar pneumonia; 36 cases
were caused by S. pneumoniae alone or concomitantly with other agents (Table
8). Blood culture was positive in four cases. Serology alone provided 32
diagnoses, while serology and blood culture were simultaneously positive in
three cases, and blood culture alone in one case (Table 14). In 66 % (23/35) of
the serologically diagnosed infections, only one test was positive, while in 34 %
(12/35) of the cases two tests were positive. All three assays were never positive
simultaneously. Fourty-six percent (16/35) of positive cases were identified by
ICs only while in 40 % (14/35) only free Ply antibodies were found.
The second series (V) included 34 children with consolidated pneumonia.
Pneumococcus was detected from blood cultures in two cases, and from lung
aspirates in 53 % (18/34) of patients, or in 65 % from those 26 of whom a
representative aspirate sample was obtained. Of all the 18 pneumococcal diagnoses
from lung aspirates, 10 were based on culture and eight were concomitantly
positive with Ply-PCR. PCR only detected eight additional cases (Table 12).
The 32 patients with only serology-proven pneumococcal pneumonia tended
to be older than those 22 in whom the agent was detected from blood culture or
lung tissue (mean 4.7 vs. 3.3. years, p = NS). The former group had lower
values for CRP (mean 144 mg/L vs. 201 mg/L, p = 0.02) (Figure 7) and ESR
(66 mm/H vs. 84 mm/H, p = 0.04) on admission. There were no significant
differences in WBC or the degree of fever (data not shown).
Table 14. Comparison of serologic methods and blood culture in pneumococcal
pneumonia (I).
Assay*

Pneumolysin antibodies (Ply-ab)

Pneumolysin immune complex (Ply-IC)
C-polysaccharide immune complex (C-PS -IC)
Blood culture
* All assays performed on 79 patients.
** Number of positive cases.

Ply-ab

12**
4
1
2

Ply-IC

C-PS -IC

4
8
7
1

1
7
0
0

Blood culture Total positive

2
1
0
1

19
20
8
4

62

Figure 7. CRP of patients with pneumococcal pneumonia diagnosed by blood culture

or lung tap vs. those detected by serology. Means are denoted with lines (p < 0.05).

8.5. Pneumococcal serotypes and antimicrobial

susceptibility (V)
Twelve invasive S. pneumoniae strains were serotyped (10 lung and two blood
isolates). The following serotypes were found: type 3, 6A, 6B, 7F, 14, 18C,
19A. Once the same serotype (18C) was detected from lung and blood.
S. pneumoniae was found only from lung in five cases, while in six cases it
was simultaneously isolated from oropharyngeal samples and blood or lung.
In one case different serotypes (19A and 23F) with different antimicrobial
sensitivities were detected from lung and oropharynx. Three blood or lung
isolates and one isolate from the oropharynx were intermediately resistant to
penicillin (MIC < 1 g/mL). Three lung strains were highly (MIC > 256 g/mL)
resistant to erythromycin.

63

9 DISCUSSION
9.1. Etiology of pneumonia and other suspected
invasive infections warranting hospitalization (I)
For accurate antimicrobial treatment, causative pathogens should be identified.
In this study, the etiology of pneumonia and other likely bacterial infections of
170 children requiring parenteral antimicrobials and hospitalization were studied
prospectively.

9.1.1. Study design

The 170 children enrolled in the study were admitted to the on-call service of
either the Aurora Hospital or the Childrens Hospital, University of Helsinki
during two time periods, 1988 - 1989 and 1991 - 1993. Aurora Hospital served
as a referral hospital for the patients of practitioners of the Health Centers of
the city of Helsinki, and as the only self-referral hospital for children in need of
hospital care living in the city of Helsinki. The Childrens Hospital was mainly
responsible for children living in areas close to Helsinki.
The physician on duty examined the patients and made the decision for the
need of parenteral antimicrobial. Enrollment was based mainly on clinical
manifestations suggestive of invasive bacterial infection. Detailed instructions
based on CRP values were also formulated for the inclusion criteria in clinically
disputable cases.
The aim of this prospective study was to collect all the patients fulfilling the
inclusion criteria. It is possible, however, that some of the patients were not
asked to participate. Furthermore, it is not known how many of the patients
refused, when invited to participate in the study. Thus, the total number of
patients who should have been enrolled is not known.
The heterogeneity of the patient material may be criticized, but the study
was planned to resemble the every-day situation of on-call service, where
various clinical manifestations have to be treated without knowledge of the
etiology. The patients included in the study are a representative sample of those
children with a suspected invasive bacterial infection who are admitted to hospital
for parenteral antimicrobials. Notably, the study does not give a reliable picture
of the etiology of childhood infections treated at home. Moreover, in this hospitalbased study, infants and young children are over-represented, and thus the results
are not directly applicable to school-aged children.

64

9.1.2. Microbiologic methods

Since few of the children had a clear focus of infection from which a representative
microbiologic sample could have been obtained, the main diagnostic method
was chosen to be serology. Paired serum samples were available from all the
patients. However, in some cases there was not enough serum for all the analyses,
which may have reduced the number of cases with identified etiology.
Interpretation of the bacteriologic results in this particular study was based on
the assumption that a significant antibody response or the presence of specific
ICs is indicative of an infection caused by the bacterium in question. However,
the sensitivity and specificity of bacterial antibody assays are not well established
for the lower respiratory infections of childhood.
There are several factors that may have influenced the serologic results of
this study. False-negative results because of diminished antibody responses may
be due to the young age of the child (Claesson et al. 1989, Nohynek et al. 1995,
Korppi and Leinonen 1998), to too short a time from the onset of infection, to
previous antimicrobial therapy, to treatment begun soon after the onset of
infection (Pichichero et al. 1987), or to too short an interval between the serum
samples. In this study, paired serum samples were obtained about two to three
weeks apart, which may have decreased the rate of positive findings especially
in chlamydial MIF (Ekman et al. 1993). Furthermore, the assays used may have
been suboptimal in respect of sensitivity. In the absence of a purified, speciesspecific protein antigen for EIA, a suboptimal whole-cell suspension of a mixture
of H. influenzae or M. catarrhalis isolates had to be used (Leinonen et al. 1981).
The pneumococcal etiology was investigated by measuring antibodies to Ply
and ICs containing IgG antibodies to Ply, and C-PS. Since many children were
too young to respond to most pneumococcal polysaccharides (Claesson et al.
1989, Korppi and Leinonen 1998), antibodies to capsular polysaccharides were
not measured. In contrast to species-specific antibodies, measurements of the
response to Ply and C-PS should theoretically detect all pneumococcal infections.
However, it is known that all the assays for detecting pneumococci have limited
sensitivity, and consequently the absence of measurement of free antibodies to
C-PS and of the response to type-specific capsular polysaccharides may have
decreased the number of pneumococcal infections detected (Korppi et al. 1993,
Korppi and Leinonen 1998, Toikka et al. 1999b, Heiskanen-Kosma 2000).
Moreover, some false- positive cases may have resulted from concomitant upper
respiratory infection (Virolainen et al. 1996, Rapola et al. 2001). Despite these
shortcomings of the specific tests and of serology in general, the assays used are
the most common standardized serologic tests currently available.
In this series, in accordance with previous studies (Nohynek et al. 1991,
Juvn et al. 2000), all viral findings, whether reached by antigen or antibody

65
assays, were considered indicative of viral infection. However, wholly convincing
evidence that a virus isolated from the upper respiratory tract is responsible for
concomitant lower respiratory infection is still mostly lacking (Hughes et al.
1969, Avila et al. 1990). Since the primary goal of this study was to search for
the bacterial etiology of infections, the latest, more expensive virologic methods
were not utilized.

9.1.3. Etiologic findings in relation to previous studies

The results of this series suggest considerable heterogeneity of the causative
agents in all the clinical manifestations studied. In all, evidence for an infectious
agent was obtained in 62 % of cases. In all entities, the agent most frequently
found was, as anticipated, S. pneumoniae, because this pathogen was searched
for with three different serologic assays. In 21 of 62 cases, there was co-infection
with another bacterium or virus. In this series, a purely viral etiology was rare,
as was to be expected from the study design; all the patients included were
hospitalized for parenteral antimicrobials. Among the patients with alveolar
pneumonia, no viral infections were found but a mixed viral-bacterial etiology
was detected in 11 % of cases, or 19 % of those with evidence of a microbiologic
etiology. However, in the sepsis-like infections (CRP 100 mg/L, fever 38.5C,
no respiratory symptoms), viruses alone were found in 19 % of the patients, and
in 28 % of those in whom the etiology was identified. These cases were caused
by adenovirus, which has already previously been shown to cause such high
CRP values (Ruuskanen et al. 1985).
Comparison of the present results with those of previous studies is difficult
because of lack of a similar study design. Most studies have addressed only one
specific manifestation. In most serology-based studies of pneumonia in
hospitalized children, the proportions with viral and with mixed viral-bacterial
etiology have been higher than in this series (Pailsey et al. 1984, Nohynek et al.
1991, Ruuskanen et al. 1992, Juvn et al. 2000). This probably depends partly
on the lack of sensitive viral serology in this study. Furthermore, in this study,
group pneumonia included only those cases with signs of alveolar pneumonia in
the radiograph. Other types of radiographic change, including probable interstitial
pneumonia, were included in the group of other respiratory infections. A finding
not in accord with other serologic studies of the etiology of pneumonia (Heiskanen-Kosma et al. 1998, 1999, Juvn et al. 2000), was that C. trachomatis
was found more commonly than C. pneumoniae. The reason for this is unknown.
One explanation could be that, even though MIF is sensitive in species-specific
diagnosis when interpreted by an experienced reader, there still might be crossreactivity with other types of Chlamydiae (Ozanne and Lefebvre 1992).

66
In this series, 66 % of cases with serologically proven pneumococcal
pneumonia were diagnosed by a single method. As in previous studies (Korppi
and Leinonen 1998, Toikka et al. 1999a, Heiskanen-Kosma 2000), detection of
specific ICs was more sensitive than measurement of the antibody responses from
paired sera. Also in line with several other serology-based studies (Nohynek et
al. 1991, Heiskanen-Kosma et al. 1998, Wubbel et al. 1999), here, in a
considerable proportion of the children, the etiology remained unknown (38 %),
despite the wide diagnostic panel used. The methodologic problems discussed
above probably explain this failure, but it is also possible that some cases were
caused by agents that were not searched for in this study, such as Bordetella
pertussis, coronavirus, and rhinoviruses (He et al. 1998, Juvn et al. 2000).

9.2. Effectiveness of antimicrobial treatment (II)

The advent of cephalosporins in the 1970s and 1980s led to major changes in
antimicrobial treatment in many countries, with increasing costs of therapy.
Cephalosporins are more active in vitro against H. influenzae and M. catarrhalis
than the commonly used penicillins. However, there is no clear evidence that
this simultaneously demonstrates clinical superiority (Klugman 1996), although,
in a series of lower respiratory infections in adults, cefuroxime led to a better
outcome than ampicillin (Pines et al. 1981).
In this study, we compared the clinical efficacies of parenteral penicillin and
cefuroxime in common childhood infections warranting hospitalization. The
group of patients was considered to be a representative sample of the previously
healthy children hospitalized for parenteral antimicrobials. Since the regimen
was randomized, no selection should have occurred between the groups. The
primary endpoint for successful treatment was uneventful recovery. Duration of
fever and need for antipyretics were not used as separate outcome measures,
because antipyretics are frequently given to children as analgesics. Although, in
about half of the patients, a bacterial agent was not proven, the clinical
presentation and laboratory values were highly suggestive of an invasive infection,
and initiation of antimicrobials would probably have been the usual practice in
most hospitals.
It may be argued that the power of the study was too low to detect a difference
if there was any. However, power calculations indicated that a total sample size
of 142 patients would disclose a 20 % difference in the effectiveness of the two
treatments. The study design, in which both the regimen used and the length of
treatment were randomized, may have caused problems when analyzing the
two main groups and when assessing the roles of these two factors in the cases

67
considered as treatment failures. Because of the small sizes of the subgroups,
statistical analysis was not done. The subgroups were, however, similar with
regard to initial characteristics, recovery, and outcome.
It may also be argued that antimicrobial resistance especially in cases of
S. pneumoniae is now more common than at the time when the study was
conducted. In Finland, the results are, however, still applicable, since the level
of resistance has remained low (Forward 1999). Even if the resistance situation
were to change, pneumococcal penicillin resistance should not be a problem in
the treatment of pneumonia and sepsis (Friedland 1995, Pallares et al. 1995,
Klugman 1996). Moreover, the rate of -lactamase producing H. influenzae
and M. catarrhalis strains has remained quite stabile during recent years,
suggesting that the efficacy of penicillin is probably quite similar to that during
the study.
The relevance of -lactam therapy in children in general may be questioned
because of the possibility of infection with M. pneumoniae or C. pneumoniae
(Ruuskanen and Mertsola 1999). Clinical failures have previously been reported
when M. pneumoniae pneumonia was treated with -lactams (Ruuskanen et al.
1992, Gendrel et al. 1997). On the other hand, in childhood pneumonia,
eradication of C. pneumoniae has occurred in children < 5 years of age during
treatment with amoxicillin-clavulanate (Harris et al. 1998). In this series, a
diagnostic increase in either Chlamydia or Mycoplasma antibodies was found
in 13 children, 12 of whom were treated successfully with parenteral -lactam.
These findings raise two questions:
1) What is the true pathogenic role of Chlamydia and Mycoplasma in these
clinical manifestations, and
2) Is the seroresponse measured caused by current specific or nonspecific
infection, or by some previous disease, by asymptomatic carriage, or by
some other factor only.
Although there has been no previous comparative study on similar infections
requiring parenteral antimicrobials in children, indirect evidence for the clinical
effectiveness and safety of the narrow spectrum antimicrobials has been obtained
from prospective and retrospective analyses of prescription habits and their
influence on the outcome. In the United Kingdom, no increase was observed in
the clinical treatment failures when several doctors in a pediatric hospital began
to treat common infections with parenteral penicillin, instead of the previously
used broader-spectrum antimicrobials (Clements et al. 2000). Furthermore, a
recent study from Uruguay showed that children with suspected bacterial
pneumonia treated with penicillin and its derivatives had a good outcome (Prez
et al. 2001).

68

9.3. Shortening of therapy (III)

At present, the tendency is to discuss which antimicrobial to use rather than to
question whether the optimum length of treatment could be shorter than it used
to be. Short regimens, when possible, would have obvious advantages in terms
of compliance and cost (Pichichero and Cohen 1997). Other potential benefits
are fewer side effects and visits by the physician, and increased patient compliance
and less trouble for the parents. Potential disadvantages, on the other hand, are
treatment failure, relapse, or recurrence, and an increased risk of complications
(Pichichero and Cohen 1997).
This study investigated shortening of the length of parenteral -lactam therapy
in lower respiratory infections and in other common invasive infections in 154
children warranting hospitalization. When either procaine penicillin or cefuroxime
was used, no difference in outcome was found among patients treated for 4 or
7 days. All the patients recovered. With longer parenteral treatment, an evident
problem with compliance was seen in this study, since 38 % of children in the
7-day group preferred to complete their medication orally at home. All the
patients who completed their randomized course either parenterally or orally
were included in the analysis, since previous findings had also suggested that
sequential antimicrobial therapy with a change from parenteral antimicrobial to
oral administration provides efficacy equivalent to a full course of parenteral
treatment (Vogel 1995). In this series, there were no differences in the outcome
of the patients whose treatment was completed parenterally and those whose
treatment was completed orally at home (data not shown). Randomizing both
the regimen and the length of treatment did not either influence the outcome.
Since the study was conducted, general treatment polices regarding the length
of parenteral therapy have changed. The mean length of parenteral administration
in the pneumonia series (V) of 34 children was only 3.5 days, although the
antimicrobial was often continued with oral regimens. Recently, oral -lactam
therapy for more than five days was shown to be associated with an increased
risk of carriage of penicillin-resistant S. pneumoniae (Guillemot et al. 1998).
Such a trend may be avoided by parenteral therapy (Klugman 1996). These
earlier findings, together with the results of this study, suggest that 4-day
parenteral -lactam therapy may be the most effective treatment in terms of
outcome, compliance, and restriction of the spread of resistant strains.

69

9.4. Lung tap in the diagnosis of pediatric

pneumonia (IV, V)
The difficulty of identifying the cause of pneumonia on clinical grounds, together
with the increasing number of bacteria resistant to the commonly used
antimicrobials has emphasized the importance of accurate etiologic diagnosis.
In the absence of commonly used sensitive gold standard method, indirect
methods for detecting the etiology are mostly not optimally validated. In pneumonia,
there is an evident need for improved diagnostics. This study tested the pertinence
of lung tap for this purpose. The aim was to obtain a sample directly from the
focus, uncontaminated by the oropharyngeal flora (Mimica et al. 1977).

9.4.1. Representativeness of the sample

This study included only patients with consolidated pneumonia. These children
are usually treated in hospital, and their disease is usually relatively severe.
Thus, the results do not give a reliable picture of childhood pneumonia in general.
In patients treated as outpatients the disease tends to be milder, the radiographic
changes less clear, and a bacterial etiology may be less frequent. Lung tap is
probably not a sensitive method for such cases, since the yield improves with
the size of the consolidation (Zalacain et al. 1995).

9.4.2. Positivity rate of lung tap

The positivity rate of a lung tap depends on the representativeness of the sample
and on the microbiologic methods used. Since the location of the consolidation
is determined from a chest radiograph and clinical signs only, there is always a
possibility that the needle does not reach the consolidation, and that consequently
the tap gives false-negative results. In this study, in accordance with a previous
series (Zalacain et al. 1995), the presence of leukocytes seen on staining was
considered indicative of a successful procedure.
Culture as such has limited sensitivity, which is known to be reduced somewhat
by pre-tap antimicrobials (Vuori-Holopainen and Peltola 2001). Furthermore,
the number of organisms present in the infectious focus may vary with the type

70
and with the phase of infection (Thomas and Parker 1920, Hammerschlag 1995).
To avoid the effect of previous antimicrobials and too few pathogens present
for culture, it may be valuable to use PCR and detection of antigens from the
lung aspirates (Boersma et al. 1991, Ruiz-Gonzles et al. 1997).
In this study, four bacterial and two viral PCR assays were used on the lung
aspirates. These assays led to 10 diagnoses not detectable by other methods.
Since PCR is extremely sensitive and does not require viable organisms, it may
cause some false-positive results. Therefore, the relevance of a positive Ply-PCR
in the absence of a simultaneous culture of pneumococcus may be questioned.
A likely explanation is that the sample contained only a minute number of bacterial
cells. Unlike previous studies of pneumonia applying Ply-PCR to blood samples
(Rudolph et al. 1993, Salo et al. 1995, Dagan et al. 1998, Toikka 1999a), in this
study, each sample was taken directly from a consolidated infectious focus.
Thus, in contrast to blood-based PCRs, false-positive results caused by nasopharyngeal carriage (Dagan et al. 1998) should be rare. Furthermore, in this
study, a positive Ply-PCR was associated well with the presence of leukocytes in
the stained samples, suggesting that the assay had a high specificity. False-positive
results caused by -hemolytic streptococcus (Kearns et al. 2000) were evidently
not a problem in this series since none of the lung aspirates grew that agent.
However, some unexpected negative results were obtained in cases with a
representative sample. Some may have occurred as a result of previous antimicrobial treatment (Dagan et al. 1998). Another possible explanation is that
the sample was taken too early (Scott and Hall 1999), and that therefore too
few pathogens were present (Thomas and Parker 1920). Finally, because the
volume of the samples was limited, no tests were made for the presence of
DNA polymerase inhibitors in PCR-negative samples (Higushi et al. 1989). In
optimal settings, for elimination of false negative results, only patients with
representative sample should be included and tests should be made for the
presence of PCR inhibitors.
Like the present results, all previous studies employing lung tap have failed
to prove the etiology in all cases (Scott and Hall 1999, Vuori-Holopainen and
Peltola 2001). This may be due in part to the problems of methodology and
sample validity discussed above. False-negative results are also obtained if
aspirate cultures are considered negative when an isolated organism does not fit
with our current knowledge of pneumonia etiology (Scott and Hall 1999). It
has also been suggested that agents that still remain to be discovered, or are
thought to be improbable pathogens may be responsible for some cases of
pneumonia without known etiology (Fang et al. 1990). In this study, M. osloensis

71
was identified once. A review of the literature removes any doubt of its pathogenic
role in pneumonia. One of the reasons why it has so seldom been identified
before is probably that few pneumonia studies are based on culture, and no
routine PCR or serologic test for that bacterium has been available.
Another hypothesis to account for cases of pneumonia of unknown etiology
is that most of them are undetected pneumococcal infections (Ruiz-Gonzales et
al. 1999). This view is based on the fact that study of lung aspirates has provided
evidence of pneumococcal etiology in cases, which remained undiagnosed by
conventional methods, and that sensitive PCR has further increased the number
of pneumococcal diagnoses. In this study, in accordance with that view, 16
pneumococcal infections unrecognized by blood culture were detected by lung
tap. Addition of other pneumococcal PCR assays, as well as antigen detection
methods, might have further increased the numbers with pneumococcal etiology
(Ruiz-Gonzales et al. 1997, Garcia et al. 1999).

9.4.3. Ethical considerations and applicability of lung tap

Invasive methods, such as lung tap or transtracheal aspiration, are seldom
considered justified, especially in developed countries, where the outcome of
pediatric pneumonia is favorable (Ruuskanen and Mertsola 1999). This view is
based on the belief that the risk of potentially harmful complications is considerable.
However, a recent review of the published literature on lung taps in children
indicates that the method is safer than has been generally thought (Vuori-Holopainen and Peltola 2001). With careful observation of the child after the
procedure, all potential adverse events requiring treatment can be dealt with.
By restricting the use of the method to a few experienced physicians, the rate of
adverse events has been decreased, while the chances of obtaining a valid sample
are increased (Zalacain et al. 1995).
Lung tap has an undoubted role in etiologic studies of pneumonia. It provides
considerable, highly specific, additional information on blood cultures, on species
and serotypes causing disease, and on antimicrobial sensitivities. It is also a
valuable methodologic tool for comparing other noninvasive diagnostic methods.
Its role may become more important in the areas where antimicrobial resistance
is increasing. In view of these findings, and of the experience gained in the
present study, we consider that lung tap is justified in well-equipped hospitals in
research settings, and apart from the etiologic studies for patients who have not
responded to the initial therapy.

72

9.5. Invasive isolates of Streptococcus

pneumoniae (V)
Identification of the pneumococcal serotypes causing most infections is critical
for the design of multivalent conjugate vaccines. Since causative serotypes vary
both geographically and temporally (Butler et al. 1995, Sniadack et al. 1995),
information is needed from several parts of the world. Pneumococci identified
from the nasopharynx have been suggested as surrogate markers for those causing
pneumonia or other forms of invasive pneumococcal disease (Lloyd-Evans et
al. 1996). In this study, some degree of correlation was found between the
oropharyngeal and the lung strains, but carriage without concomitant detection
from the lung was common, and, in one case, the lung and oropharynx yielded
different types with different antimicrobial sensitivities. Furthermore, in five
cases, pneumococcus was isolated from the lung only. Current heptavalent
conjugate vaccine (Rubin 2000) would have prevented only five of the 11 cases
with culture-positive pneumococcal pneumonia detected in this study. In future,
type-specific pneumococcal PCR may provide additional information about the
serotypes of the strains that are detectable only by sensitive PCR.
Although penicillin resistance has gained much attention worldwide, its role
in Finland has remained a minor one (Finres 1999). Our sample is small, and no
definitive conclusions should be drawn. However, no fully penicillin-resistant strains
were found; meanwhile, three isolates were resistant (MIC > 256 g/mL) to
erythromycin. This is in accord with current resistance figures, which show that,
in the Helsinki area, up to 12 % of S. pneumoniae strains are resistant to erythromycin (Dr M. Vaara, personal communication, 2001). This finding may reflect the
current trend for prescribing macrolides in childhood respiratory infections.

9.6. General discussion and future

considerations
The morbidity and mortality due to pneumonia have evidently been influenced
by antimicrobials (Hui et al. 1997, Yang et al. 2001). The role of the new,
widely-marketed, broad-spectrum agents, however, is controversial. In a retrospective analysis of mortality from pneumonia in US children over the past halfcentury, the steepest decline in mortality occurred in the late 1940s, probably as
a result of penicillin. A series of new antimicrobials has been introduced since
then, but no association has been found between further reductions in mortality
and the introduction of these agents (Dowell et al. 2000).

73
In many nonindustrialized countries, high infant mortality is accepted by
parents as a fact of life and is one reason for having large families. Marked
reductions in childhood mortality due to pneumonia could probably be achieved
by expanding immunization programs and by better diagnostic practices and by
early and appropriate management (Vaahtera et al. 2000). Inexpensive and easily
administered medicaments would be especially useful in such settings (Kaplan
2000). The present results with penicillin and with short courses of antimicrobials
cannot directly be applied in the circumstances in developing countries, but
the experience may encourage the colleagues to conduct similar studies in
those countries.
In the industrialized world, the main reason for the indiscriminate use of
wide-spectrum antimicrobials is concern about bacteria potentially insensitive
to narrow-spectrum regimens. Besides comparative studies on the clinical efficacy
of different antimicrobials, reliable information on the etiology is also required
for formulation of relevant guidelines for prescribing doctors (Clements et al.
2000). On the basis of the present results, S. pneumoniae seem to be the most
common agent causing childhood pneumonia and other invasive bacterial
infections. Since the proportion of penicillin resistant strains has remained low
in our circumstances, penicillin can be considered as a treatment of choice in
these infections.
Priorities for future research on pediatric pneumonia include at least the
following:
1) Determination of the sensitivity and specificity of commonly used, noninvasive
methods in relation to lung tap, a specific method for pneumonia diagnostics.
2) Validation of various serologic methods in the light of the information
obtained by lung tap.
3) Collecting data on pneumococcal serotypes obtained from lung tissue for
assessing the relevance of conjugate vaccine in the prevention of pneumonia.
4) Re-evaluation of the relevance of acute-phase parameters and chest
radiographs for the etiologic diagnostics with respect to pathogens identified
directly from the infectious focus.

74

10 SUMMARY AND CONCLUSIONS

Although most cases of childhood community-acquired pneumonia and other
common infections can be treated adequately with empirical medication, the
etiologic diagnosis is important for seriously ill patients, and for those who have
failed to respond to standard therapy. Microbiologic diagnosis also enables the
clinician to direct the treatment against specific pathogens and to avoid
unnecessary medication. Furthermore, etiologic data are needed for treatment
guidelines and for development of vaccines.
In the present study, the etiology and treatment of childhood pneumonia,
other respiratory infections, sepsis-like infections, and other acute infections
requiring parenteral antimicrobials were investigated. Meningitis, urinary tract
infections, and osteoarticular infections were excluded. Mainly serologic evidence
for a microbial etiology was found in 62 % of children. The distribution of
pathogens was heterogeneous, the dominant agent in all manifestations being
S. pneumoniae. These infections were cured by parenteral penicillin as well as
by cefuroxime, and the 4-day parenteral -lactam proved as effective as the 7-day
therapy. Hence, in most common childhood suspected invasive infections, short
courses of narrow-spectrum parenteral -lactam antimicrobials seem justified.
The accuracy of the microbiologic diagnostics in childhood consolidated
pneumonia requiring hospitalization was significantly improved by using a lung
tap. Culture of the aspirate yielded the diagnosis in 32 % of 34 cases, but when
modern microbiologic methods were applied, the etiology was identified in 59 %
of all cases, and in 69 % of those in which a representative lung-tap specimen
was obtained. In consolidated pneumonia, the leading pathogen was S. pneumoniae.
However, several other pathogens that were not predictable from the clinical
signs or laboratory parameters of the patient were also found. The advantages
of lung tap are the high microbiologic yield and a relatively low risk of adverse
events. We consider lung tap applicable in hospital-based etiologic studies, and
apart from research, in circumstances in which the need to identify the causative
agent outweighs the modest risks of the procedure.

75

11 ACKNOWLEDGMENTS
I wish to thank Emeritus Professor Jaakko Perheentupa, the former Head of the
Hospital for Children and Adolescents, University of Helsinki, and Professor
Mikael Knip, the current Head of the Hospital for Children and Adolescents,
for providing excellent research facilities.
I am most grateful to my supervisor, Professor Heikki Peltola, for providing
me with the study materials and facilities required and, more importantly, for
teaching me all about pediatric infectious diseases, clinical research, science,
and life in general. His vast experience and global view of infectious diseases,
his innovative ideas and continuous energy, and, most of all, his never-failing
encouragement and support have made this thesis possible.
I am grateful to all my collaborators, whose help has been invaluable during
this work. I wish to express my warmest gratitude to Markku Kallio, MD, for
his enthusiastic guidance and advice in work as well as in personal life. His
unlimited energy and his continuous disinterested support and help have been
invaluable. I also warmly thank Eeva Salo, MD, and Docent Harri Saxn for
collecting the patients, for critical comments on the manuscripts, and for giving
much advice about science, as well as for keeping my feet on the ground.
Professor Martti Vaara, Professor Maija Leinonen, and Docent Klaus Hedman
deserve special thanks for their vast experience and valuable advice concerning
our projects and manuscripts. I also want to thank all the other co-authors and
the SE-TU Study Group, for good and fruitful collaboration.
An essential part of this study has been collaboration with the nurses,
laboratory personnel and other staff in the participating hospitals and laboratories.
I owe my warmest thanks to all of them. Special thanks go to the personnel of
the on-call service of Hospital for Children and Adolescents for whom our KETU study has sometimes caused plenty of extra work, and to Ms Raija Lahdenper of Laboratory Diagnostics, Helsinki University Central Hospital, for all
the advice, e-mails, and phone calls during this project. Without her initiative,
experience, and positive attitude, many things would have been much more
complicated. I also want to express my gratitude to the patients and their parents
for their co-operation, without which this study would not have been
accomplished.
I would like to thank the official reviewers of this thesis, Professor Jussi
Mertsola and Professor Matti Korppi, for their positive attitude, constructive
criticism, and good advice. Mrs Jean Margaret Perttunen revised the language
of this thesis, for which I am very grateful. I also highly appreciate Ms Terttu
Halmes work on the layout of the thesis.
I also acknowledge, with thanks, financial support of Helsinki University
Biomedical Graduate School, the EVO Funds of Helsinki University Central

76
Hospital, the Paulo Foundation, the Vin and Laina Kivi Foundation, the
Research Foundation of Orion Corporation, the Finnish Medical Foundation,
and Pfeizer Ltd. for this work.
Several colleagues and friends have given strong support during the project,
which I highly appreciate. Tarja Heiskanen-Kosma, MD, has given valuable
criticism and unlimited support while I was writing the thesis. Annamari Mkel,
MD and Pivi Sormunen, MD have started from the same chick phase with
me and shared the process of how to do clinical research under the supervision
of Professor Heikki Peltola. Mrs. Riitta Meurman is warmly thanked for mental
support, for continuous interest in my family life, and for technical assistance
with the figures and tables. Colleagues Johanna Paronen and Kirsta Erkkil are
warmly thanked for their friendship and for sharing the continuous difficulties
of being a researcher and a good mother.
My parents, Tuire and Arto, are thanked for their sympathy and support, and
for implanting in me the idea of the importance of meaningful work. My warmest
thanks go to my sister Elisa. Her support, understanding, and friendship has so
many times encouraged me to continue. I am also grateful to my parents-inlaw, Pirkko and Martti, for all the support I have received.
My deepest gratitude goes to Juha, my dear husband. His endless love,
support, encouragement, and understanding over the years have been invaluable.
I could not have coped without him. And finally, I want to thank our dear
daughter Ronja, who provides so much joy every day. You two, my beloved
family, make my life worthwhile.

Helsinki, August 2001

77

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