Previous Article | Next Article 
Journal of Clinical Microbiology, March 1999, p. 633-637, Vol. 37, No. 3
Department of
Pediatrics1 and
Department of Clinical
Microbiology,
Received 13 May 1998/Returned for modification 22 October
1998/Accepted 18 November 1998
Blood-based pneumolysin PCR was compared to blood culture and
detection of pneumolysin immune complexes, as well as to detection of antibodies to pneumolysin and to C polysaccharide, in the
diagnosis of pneumococcal infection in 75 febrile
children. Invasive pneumococcal infection was suspected on clinical
grounds in 67 of the febrile children, and viral infection was
suspected on clinical grounds in 8 of the febrile children. In
addition, 15 healthy persons were examined to test the specificity of
the PCR assay. Plasma, serum, and leukocyte fractions were analyzed by
PCR. The combination of all test results led to the diagnosis of
pneumococcal infection in 25 patients. Pneumolysin PCR was positive in
44% of these children, an increase occurred in the pneumolysin
antibodies in 39% and in the C polysaccharide antibodies in 30% of
the patients; pneumolysin immune complexes were found in
convalescent serum in 30%, pneumolysin immune complexes occurred
in acute-phase serum samples in 16%, and a positive blood culture was
found in 20% of the patients. None of the healthy controls had
positive results by PCR. The results suggest that the diagnosis of
Streptococcus pneumoniae infection from blood samples
necessitates the use of several different assays. Pneumolysin PCR was
the most sensitive assay, but its clinical value is reduced by the fact
that three blood fractions are needed.
Streptococcus
pneumoniae is the predominant causative agent of childhood
invasive bacterial infection in countries where infections caused by
Haemophilus influenzae type b are eliminated by vaccinations (12, 24). The main clinical syndromes associated with
invasive pneumococcal infection are occult bacteremia, pneumonia,
meningitis, peritonitis, periorbital cellulitis, and septic arthritis
(6, 7). One study suggests that if a child with occult
pneumococcal bacteremia is not treated with antibiotics, there is a 6%
risk for meningitis (2).
The differentiation of invasive pneumococcal infection from other
febrile illnesses is difficult in the early phase of the disease.
Children aged 3 to 36 months with fever of A definitive diagnosis of invasive pneumococcal infection requires the
isolation of S. pneumoniae from normally sterile sites such
as the blood, lungs, pleural fluid, cerebrospinal fluid, or synovial
fluid. Recently, antibody assays for S. pneumoniae, as well
as measurement of circulating immune complexes, have proved useful in
the study of the role of S. pneumoniae in the etiology of
acute lower-respiratory-tract infections in young children (16,
20).
We compared pneumolysin PCR, blood culture, and detection of
pneumolysin immune complexes, as well as of antibodies to pneumolysin and to C polysaccharide, for the diagnosis of invasive pneumococcal infection in febrile children.
Patients.
Febrile children admitted during a 5-month period
(beginning August 1996) to the Department of Pediatrics, Turku
University Hospital, were enrolled in the study. The inclusion criteria
were: a serum C-reactive protein (CRP) value of Peripheral blood samples.
Blood samples were obtained during
routine diagnostic evaluation. In 89% of cases, the samples for PCR
and the samples for detection of antibodies and immune complexes were
taken within 24 h after admission. From each patient, 3 ml of
blood was collected for the serum sample, and 2 to 9 ml (mean, 6 ml) of
blood was collected in tubes containing EDTA. One milliliter of the
EDTA blood was used for separation of the plasma, and the rest was diluted with Hank's buffered saline with sodium bicarbonate at a ratio
of 1:1. The WBC fraction was separated from the diluted blood by
density centrifugation (Ficoll; [Pharmacia Biotech, Uppsala, Sweden]
and Histopaque 1119 [Sigma Diagnostics, St. Louis, Mo.]). The layers
of mononuclear cells and granulocytes were aspirated and then washed
with phosphate-buffered saline (400 g for 10 min) in a total volume of
40 ml.
Purification of DNA from WBC, plasma, and serum.
The serum
samples were stored at Pneumolysin PCR.
The PCR amplifications were done with a
programmable thermal cycler (GeneAmp PCR system 9600; Perkin-Elmer,
Norwalk, Conn.) in a 50-µl volume with pneumolysin primers
(25). The outer primers Ia
(5'-ATTTCTGTAACAGCTACCAACGA-3') and Ib
(5'-GAATTCCCTGTCTTTTCAAAGTC-3') amplified a 348-bp region of
the pneumolysin gene, and the inner primers IIa
(5'-CCCACTTCTTCTTGCGGTTGA-3') and IIb
(5'-TGAGCCGTTATTTTTTCATACTG-3') amplified a 208-bp region.
The reaction mixture contained 10 mM Tris-HCl (pH 8.8), 1.5 mM
MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 µM
deoxyribonucleotides, 50 pmol of primers, 1.0 U of DNA polymerase
(Dynazyme; Finnzymes, Espoo, Finland), a drop of mineral oil, and
various amounts of DNA (1:1 and 1:10 dilutions) extracted from WBC
fraction, plasma, and serum specimens (5 µl). The amplifications were
repeated 40 times as follows: 30 s at 94°C for denaturation, 30 s at 56°C for annealing, and 30 s at 72°C for
extension. Nested amplifications were carried out as for the
first-round PCR. A pneumococcal DNA preparation (ATCC 49619) was used
as a positive control. At every amplification, a negative control was
included. The PCR products were stored at 4°C prior to analysis by
agarose gel electrophoresis.
Blood culture.
Blood samples for cultures were obtained
prior to the initiation of antimicrobial therapy. The blood culture
bottles (Dupont Isolator system; Merck, Darmstadt, Germany) were
inoculated with blood obtained by peripheral venipuncture from 66 children upon admission; 59 of the 67 patients in the study group and 7 of the 8 children in the comparison group were tested.
Determination of antibodies to pneumolysin and to C
polysaccharide and pneumolysin immune complexes.
Paired serum
specimens were obtained from 54 patients: 50 of the 67 patients in the
study group and 4 of the 8 patients in the comparison group. The
acute-phase samples were drawn from all patients at the first visit,
and the convalescent-phase samples were collected 34 ± 20 (mean ± standard deviation [SD]) days later. These samples were
used for the determination of pneumolysin antibodies, C polysaccharide
antibodies, and pneumolysin immune complexes. A twofold or greater rise
in antibody titer to pneumolysin or C polysaccharide in paired sera or
the presence of pneumolysin-specific immune complexes in any serum
sample were the serological criteria used to determine the presence of
pneumococcal infection (13, 16, 18).
In vitro sensitivity of PCR.
The sensitivity of the PCR was
evaluated by using pneumococcal DNA as a target at 10-fold dilutions.
Both in the first amplification round and in the nested PCR,
sensitivity was 90 fg as detected by visualization of PCR products of
the expected 208-bp length by agarose gel electrophoresis. Southern
hybridization of the PCR products caused a 10-fold increase in the
sensitivity of the PCR reactions. When whole pneumococci were used as a
target in serial dilutions, the sensitivity of the assay was 10 CFU and 1 CFU after Southern hybridization of the PCR product.
Patient characteristics.
The clinical diagnosis and numbers of
pediatric patients fulfilling the study inclusion criteria
(n = 67) were as follows: pneumonia, 39; fever without
infection focus, 16; acute respiratory tract infection, 5;
pneumococcemia, 2; meningitis with blood culture confirmed S. pneumoniae septicemia, 1; periorbital cellulitis with blood
culture confirmed S. pneumoniae septicemia, 1; periorbital cellulitis, 1; Bacteroides fragilis septicemia, 1; and acute
tonsillitis, 1. All patients were treated with antibiotics. The
patients in the comparison group (n = 8) received the
following diagnoses: interstitial pneumonia, 4 patients; acute
respiratory tract infection, 2 patients; parotitis, 1 patient; and
aseptic meningitis, 1 patient.
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Pneumolysin PCR-Based Diagnosis of Invasive
Pneumococcal Infection in Children
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
39°C and a leukocyte
(WBC) count of 15 × 109/liter should be
suspected to have invasive bacterial infection (1,
9). These signs are, however, also common in children with
viral infections (23).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
100 mg/liter, a WBC count of 15 × 109/liter, or alveolar
pneumonia. Sixty-nine patients fulfilled the criteria, and the
final number of patients with suspected invasive pneumococcal infection
was 67 after the exclusion of two patients with urinary tract
infection. In addition, blood samples from eight febrile children with
a virus-type infection (well-appearing children with a body temperature
of <39.0°C, a CRP value of <80 mg/liter, and a WBC of <15 × 109/liter) were included for comparison, and blood from
15 healthy persons was examined to test the specificity of the PCR assay.
20°C before isolation of DNA. DNA was
isolated from plasma and WBCs within 1 h in 41% of cases and
within 24 h in 75% of cases. The WBC fraction was centrifuged for
10 s, and the pellet was suspended with 200 µl of
gamma-irradiated water. The WBC fraction was incubated for 10 min at
94°C before proteinase K (2 µl, 10 mg/ml; Boehringer Mannheim,
Mannheim, Germany) treatment. After incubation for 1 h or
overnight at 56°C, the same protocol was used for 200 µl of plasma,
serum, and WBC. First, 300 µl of sodium dodecyl sulfate (SDS)
containing 0.1 M NaOH, 2 M NaCl, and 0.5% SDS was added to the
suspension, which was then incubated for 15 min at 95°C. Then, 200 µl of 0.1 M Tris-HCl (pH 8.0) was added. DNA was extracted with
phenol, precipitated with ethanol, and dissolved in 25 µl of
Tris-EDTA.
70°C.
RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
PCR-positive peripheral blood specimens.
There were 12 PCR-positive peripheral blood samples. Nine were found by
visualization of the PCR products by agarose gel electrophoresis: three
from the WBC fraction, 3 from the plasma samples, and three from the
serum samples (Table 1). All positive
results were confirmed by Southern hybridization. In addition, three
samples were positive after Southern hybridization of the PCR product.
Two of these were positive WBC fractions, and one was a positive plasma
sample. In one patient, the infection was diagnosed from the plasma
sample by agarose gel electrophoresis and, additionally, from the WBC fraction by Southern hybridization. Thus, 11 patients were found to
have positive samples by PCR. None of the patient samples from the
comparison group and none of the samples of the healthy controls were
found to be positive by PCR.
|
Blood culture-positive specimens. S. pneumoniae was isolated from blood culture in four patients. The densities of bacteria in the four cultures were <1, 1, 2, and 200 bacteria/ml. The first case was diagnosed by pneumolysin PCR, as well as by an increase in pneumolysin and C polysaccharide antibodies. The second case was not positive by any other method. The third case was determined to be positive by detection of pneumolysin immune complexes in the convalescent serum, and the fourth case was found to be positive by pneumolysin PCR. The two patients with negative PCR results had been on antibiotic treatment for 2 and 5 days before the blood samples for PCR were taken, and the two patients with positive PCR results had been on antibiotic treatment for 1 day.
Positive results in pneumolysin immune complexes or diagnostic
increases in pneumolysin or C polysaccharide antibody titers.
Pneumococcal infection was diagnosed from an increase in pneumolysin or
C polysaccharide antibodies or by the presence of pneumolysin immune
complexes in 19 patients. Four of these were also diagnosed by PCR, and
two were diagnosed by blood culture (Table
2). None of the patients with pneumolysin
immune complexes in their acute-phase serum samples had positive
results by pneumolysin PCR or diagnostic increases in pneumolysin
antibody titers. Four patients had pneumolysin immune complexes in
their sera both in the acute and the convalescent phases. Two of these
children were from the comparison group with interstitial pneumonia
with a WBC count of <15 × 109/liter and a CRP value
of <80 mg/liter.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
This study shows that the diagnosis of invasive pneumococcal infection in children necessitates the use of a combination of several tests. Pneumococcal infection was diagnosed in 25 of the patients in this study group and in 2 patients in the comparison group. Of the 25, 44% could be diagnosed by pneumolysin PCR, 39% by an increase in pneumolysin antibodies, 30% by an increase in C polysaccharide antibodies, 30% by the presence of pneumolysin immune complexes in convalescent-phase serum samples, 20% by blood culture, and 16% by the presence of pneumolysin immune complexes in acute-phase serum samples.
Only a few studies have been published in which PCR was used to diagnose invasive pneumococcal infection. Rudolph et al. (21) studied 16 adults with culture-proven pneumococcal bacteremia by using nested PCR, with primers designed from pneumolysin and autolysin genes. In vitro sensitivity was 10 fg or 200 CFU for whole bacteria and 20 CFU for buffy-coat samples. The sensitivity of the assay when buffy-coat fraction samples from eight blood culture-positive patients were studied was 75% with pneumolysin and 63% with autolysin primers. When eight whole-blood specimens were tested, the sensitivity was 37.5% with either set of primers, and the specificity of the assay was 93%. In another study from Gambia, 25 adults with suspected pneumonia were examined by PCR also with primers detecting the autolysin gene sequence (10). In vitro sensitivity was 50 fg or 3 CFU. The samples were first cultured and the DNA for PCR analysis was extracted from blood culture bottles either 48 h after inoculation, if positive growth was recorded, or after 7 days, when the culture bottles were discarded. S. pneumoniae was isolated from blood cultures in 12 patients. In four patients, PCR was positive with supernatants from both paired culture bottles, whereas pneumococci were cultured from only one. Salo and coworkers (25) studied 20 serum samples from adult patients with blood culture-confirmed acute pneumococcal pneumonia. These authors used a nested PCR method with primers similar to those used in the present study designed from the pneumolysin gene. The in vitro sensitivity was 24 fg (10 bacterial equivalents). All 20 samples were positive by PCR. To assess clinical specificity, 100 serum specimens from healthy adults were tested and 94 were found to be negative (specificity, 94%). Zhang and coworkers (26) used whole-blood PCR to study 36 pediatric patients with suspected bacteremia. Their primers and probe were derived from the penicillin-binding protein 2B gene. The in vitro sensitivity was 100 fg or 1 CFU. Four of five blood culture-positive patients were diagnosed, as well as five additional cases from the 31 culture-negative samples. Dagan and coworkers (4) undertook a prospective study to evaluate the accuracy of pneumolysin PCR of serum for the detection of pneumococcal infections in children. The in vitro sensitivity of the pneumolysin PCR assay was 10 CFU, whereas the clinical sensitivity of blood and cerebrospinal fluid culture-positive samples from 13 patients was 100%. The positivity rates for patients with lobar or segmental pneumonia or acute otitis media and for healthy controls were 38, 44, and 17%, respectively. The results indicated that although pneumolysin PCR was sensitive, it was not very useful for the detection of deep-seated pneumococcal infections because a high rate of positivity was seen in the controls. It should be noted that the positive results by agarose gel electrophoresis in the present study were not confirmed by Southern hybridization. In our study, 69 serum, plasma, or WBC fraction samples from patients with a virus-type infection or from healthy controls were tested by PCR, and none of them were found to be positive, indicating a good specificity and a lack of contamination in our PCR procedure. Our problem was sensitivity rather than specificity, because we found only 12 PCR-positive samples among the 201 serum, plasma, or WBC samples tested.
Several factors may limit the sensitivity of PCR with samples from the peripheral blood in a clinical setting. First, the density of S. pneumoniae in the bloodstream is often low. In the Turku University Hospital, S. pneumoniae was isolated from blood cultures of 22 children during 1994 to May 1997. Of these, 23% had <1 bacteria/ml, 27% had 1 to 9 bacteria/ml, 23% had 10 to 100 bacteria/ml, and 28% had >100 bacteria/ml. This indicates that the sensitivity of the PCR may not be high enough for all blood culture-positive cases. The second limitation of PCR with blood specimens is the inhibition of DNA polymerase by porphyrin compounds (11). This decreases the sensitivity of the PCR in clinical samples compared to in vitro conditions. Moreover, antibiotic treatment before sampling decreases the yield of positive findings by PCR. Dagan and coworkers did not detect pneumococcal DNA in the serum 48 h after the initiation of antibiotic treatment (4). In our study, the two blood culture-positive patients who were pneumolysin PCR negative had been on antibiotic treatment for 2 and 5 days before the samples for PCR were taken.
Blood culture is only seldom positive in children with pneumococcal pneumonia in developed countries (3, 8, 22). Therefore, various antibody assays and the detection of pneumococcal immune complexes have been used to study the etiology of pneumonia. The diagnostic methods have shown the following sensitivities in children: pneumococcal antigen in acute serum, 33 to 39%; antibodies to type-specific capsular polysaccharides, 32 to 37%; pneumolysin antibodies, 7 to 30%; C polysaccharide antibodies, 12 to 15%; and pneumococcal antigen in an acute-phase urine sample, 2 to 5% (14, 15). Korppi and Leinonen (16) found diagnostic levels of immune complexes in nearly one-half of the pneumococcal pneumonia cases diagnosed by antigen, free antibody, or circulating immune complexes. The assays, also used in the present study, have been validated in healthy children and in young adults with a common cold; a positive result or a diagnostic rise in titers between paired sera have been present in <1% by the above criteria also used in this study (17, 18, 19).
Our study may well underestimate the value of blood culture in the detection of invasive pneumococcal infection. Isaacman et al. (12) found that a single small-volume blood culture fails to identify a significant proportion of children with bacteremia. Differences occur between blood culture methods used to detect bacteremia and fungemia. The isolator system used in our hospital has been found to be less sensitive than the Bactec system for the detection of bacteremia (5, 12). Our study is also limited by the fact that the samples for pneumolysin PCR and the samples for the detection of antibodies and immune complexes were not regularly taken before antibiotic treatment, as were the samples for blood cultures. Therefore, the results of the different assays are not fully comparable. None of the samples from patients in the comparison group or from controls were positive by pneumolysin PCR. However, pneumolysin PCR was positive with the WBC fraction of a 4-year-old girl with unilateral tonsillitis. Although this patient may have had invasive pneumococcal infection, we cannot exclude the possibility that this PCR result could also be a false-positive finding, especially because none of the other pneumococcal tests confirmed the pneumococcal etiology.
The recent apparent increase of penicillin-resistant pneumococcal infections has emphasized the necessity of accurate clinical diagnosis and laboratory confirmation of pneumococcal infections. In this study, several diagnostic methods, including blood culture, detection of pneumolysin immune complexes and antibodies to pneumolysin and to C polysaccharide, and pneumolysin PCR, were compared in the diagnosis of invasive pneumococcal infection. We conclude that a combination of several methods is needed for the detection for invasive pneumococcal infection. Pneumolysin PCR was the most sensitive assay tested and increased the number of patients with a microbiological diagnosis of pneumococcal infection. However, for optimal sensitivity, several blood fractions should be tested and this is laborious and expensive. We conclude that pneumolysin PCR is currently not a feasible method for routine diagnostics of invasive pneumococcal infection in children.
| |
ACKNOWLEDGMENTS |
|---|
We thank Tiina Haarala and Merja Mikkola for skillful technical assistance and Timo Hurme for providing samples from healthy children.
This work was supported by the Academy of Finland, the Turku University Foundation, and the Finnish Foundation for Pediatric Research.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Research Unit, Department of Pediatrics, Turku University Hospital, Vähä Hämeenkatu 1 A 3, FIN-20500 Turku, Finland. Phone: 358-2-261-2486. Fax: 358-2-261-1485. E-mail: pia.toikka{at}utu.fi.
Present address: Stanford University School of Medicine, Dept. of
Microbiology and Immunology, PAVAHCS, Palo Alto, CA 94304.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
| 1. |
Baraff, L. J.,
J. W. Bass,
G. R. Fleisher,
J. O. Klein,
G. H. McCracken, Jr.,
K. R. Powell, and D. L. Schriger.
1993.
Practice guideline for the management of infants and children 0 to 36 months of age with fever without source.
Pediatrics
92:1-12 |
| 2. |
Baraff, L. J.,
S. Oslund, and M. Prather.
1993.
Effect of antibiotic therapy and etiologic microorganism on the risk of bacterial meningitis in children with occult bacteremia.
Pediatrics
92:140-143 |
| 3. | Claesson, B. A., B. Trollfors, I. Brolin, M. Granstrom, J. Henrichsen, U. Jodal, P. Juto, I. Kallings, K. Kanclerski, T. Lagergard, L. Steinwall, and O. Strannegard. 1989. Etiology of community-acquired pneumonia in children based on antibody responses to bacterial and viral antigens. Pediatr. Infect. Dis. J. 8:856-862[Medline]. |
| 4. |
Dagan, R.,
O. Shriker,
I. Hazan,
E. Leibovitz,
D. Greenberg,
F. Schlaffer, and R. Levy.
1998.
Prospective study to determine clinical relevance of detection of pneumococcal DNA in sera of children by PCR.
J. Clin. Microbiol.
36:669-673 |
| 5. | Eisenach, K., J. Dyke, M. Boehme, B. Johnson, and M. B. Cook. 1992. Pediatric blood culture evaluation of the Bactec PEDS Plus and DuPont Isolator 1.5 systems. Diagn. Microbiol. Infect. Dis. 15:225-231[Medline]. |
| 6. |
Eskola, J.,
A. Takala,
E. Kela,
E. Pekkanen,
R. Kalliokoski, and M. Leinonen.
1992.
Epidemiology of invasive pneumococcal infections in children in Finland.
JAMA
268:3323-3327 |
| 7. | Frankel, R. E., M. Virata, C. Hardalo, F. L. Altice, and G. Friedland. 1996. Invasive pneumococcal disease: clinical features, serotypes, and antimicrobial resistance patterns in cases involving patients with and without human immunodeficiency virus infection. Clin. Infect. Dis. 23:577-584[Medline]. |
| 8. | Gendrel, D., J. Raymond, F. Moulin, J. L. Iniguez, S. Ravilly, F. Habib, P. Lebon, and G. Kalifa. 1997. Etiology and response to antibiotic therapy of community-acquired pneumonia in French children. Eur. J. Clin. Microbiol. Infect. Dis. 16:388-391[Medline]. |
| 9. | Gombos, M. M., R. S. Bienkowski, R. F. Gochman, and H. H. Billet. 1998. The absolute neutrophil count. Is it the best indicator for occult bacteremia in infants? Am. J. Clin. Pathol. 109:221-225[Medline]. |
| 10. |
Hassan-King, M.,
I. Baldeh,
S. Ousman,
A. Falade, and B. Greenwood.
1994.
Detection of Streptococcus pneumoniae DNA in blood cultures by PCR.
J. Clin. Microbiol.
32:1721-1724 |
| 11. | Higuchi, R. 1989. Simple and rapid preparation of samples for PCR, p. 31-38. In H. A. Erlich (ed.), PCR technology: principles and applications for DNA amplification. Stockton Press, New York, N.Y. |
| 12. | Isaacman, D. J., R. B. Karasic, E. A. Reynolds, and S. L. Kost. 1996. Effect of number of blood cultures and volume of blood on detection of bacteremia in children. J. Pediatr. 128:190-195[Medline]. |
| 13. | Jalonen, E., J. C. Paton, M. Koskela, Y. Kerttula, and M. Leinonen. 1989. Measurement of antibody responses to pneumolysin: a promising method for the presumptive etiological diagnosis of pneumococcal pneumonia. J. Infect. 19:127-134[Medline]. |
| 14. | Korppi, M., T. Heiskanen-Kosma, E. Jalonen, P. Saikku, M. Leinonen, P. Halonen, and P. H. Mäkelä. 1993. Aetiology of community-acquired pneumonia in children treated in hospital. Eur. J. Pediatr. 152:24-30[Medline]. |
| 15. | Korppi, M., M. Koskela, E. Jalonen, and M. Leinonen. 1992. Serologically indicated pneumococcal respiratory infection in children. Scand. J. Infect. Dis. 24:437-443[Medline]. |
| 16. | Korppi, M., and M. Leinonen. 1997. Pneumococcal pneumonia in children; new data from circulating immune complexes. Eur. J. Pediatr. 156:341-342[Medline]. |
| 17. | Korppi, M., M. Leinonen, M. Koskela, M. Mäkelä, and K. Launiala. 1989. Bacterial coinfection in children hospitalized with respiratory syncytial virus infections. Pediatr. Infect. Dis. J. 8:687-692[Medline]. |
| 18. |
Leinonen, M.,
H. Syrjälä,
E. Jalonen,
P. Kujala, and E. Herva.
1990.
Demonstration of pneumolysin antibodies in circulating immune complexes a new diagnostic method for pneumococcal pneumonia.
Serodiagn. Immunother. Infect. Dis.
4:451-458.
|
| 19. |
Mäkelä, M.,
T. Puhakka,
O. Ruuskanen,
M. Leinonen,
P. Saikku,
M. Kimpimäki,
S. Blomqvist,
T. Hyypiä, and P. Arstila.
1998.
Viruses and bacteria in the etiology of the common cold.
J. Clin. Microbiol.
36:539-542 |
| 20. | Nohynek, H., J. Eskola, M. Kleemola, E. Jalonen, P. Saikku, and M. Leinonen. 1995. Bacterial antibody assays in the diagnosis of acute lower respiratory tract infection in children. Pediatr. Infect. Dis. J. 14:478-484[Medline]. |
| 21. |
Rudolph, K. M.,
A. J. Parkinson,
C. M. Black, and L. W. Mayer.
1993.
Evaluation of polymerase chain reaction for diagnosis of pneumococcal pneumonia.
J. Clin. Microbiol.
31:2661-2666 |
| 22. | Ruuskanen, O., and J. Mertsola. Childhood community-acquired pneumonia. Semin. Infect. Dis., in press. |
| 23. | Ruuskanen, O., A. Putto, H. Sarkkinen, O. Meurman, and K. Irjala. 1985. C-reactive protein in respiratory virus infections. J. Pediatr. 107:97-100[Medline]. |
| 24. | Saarinen, M., A. K. Takala, E. Koskenniemi, E. Kela, P. R. Rönnberg, E. Pekkanen, P. Kiiski, and J. Eskola. 1995. Spectrum of 2836 cases of invasive bacterial or fungal infections in children: results of prospective nationwide five-year surveillance in Finland. Finnish Pediatric Invasive Infection Study Group. Clin. Infect. Dis. 21:1134-1144[Medline]. |
| 25. | Salo, P., A. Örtqvist, and M. Leinonen. 1995. Diagnosis of bacteremic pneumococcal pneumonia by amplification of pneumolysin gene fragment in serum. J. Infect. Dis. 171:479-482[Medline]. |
| 26. | Zhang, Y., D. J. Isaacman, R. M. Wadowsky, J. Rydquist-White, J. C. Post, and G. D. Erlich. 1995. Detection of Streptococcus pneumoniae in whole blood by PCR. J. Clin. Microbiol. 33:596-601[Abstract]. |
Journal of Clinical Microbiology, March 1999, p. 633-637, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Findlow, H., Laher, G., Balmer, P., Broughton, C., Carrol, E. D., Borrow, R.
(2009). Competitive Inhibition Flow Analysis Assay for the Non-Culture-Based Detection and Serotyping of Pneumococcal Capsular Polysaccharide. CVI
16: 222-229
[Abstract] [Full Text] -
Hanage, W. P., Kaijalainen, T., Herva, E., Saukkoriipi, A., Syrjanen, R., Spratt, B. G.
(2005). Using Multilocus Sequence Data To Define the Pneumococcus. J. Bacteriol.
187: 6223-6230
[Abstract] [Full Text] -
Arbique, J. C., Poyart, C., Trieu-Cuot, P., Quesne, G., Carvalho, M. d. G. S., Steigerwalt, A. G., Morey, R. E., Jackson, D., Davidson, R. J., Facklam, R. R.
(2004). Accuracy of Phenotypic and Genotypic Testing for Identification of Streptococcus pneumoniae and Description of Streptococcus pseudopneumoniae sp. nov.. J. Clin. Microbiol.
42: 4686-4696
[Abstract] [Full Text] -
Sheppard, C. L., Harrison, T. G., Morris, R., Hogan, A., George, R. C.
(2004). Autolysin-targeted LightCycler assay including internal process control for detection of Streptococcus pneumoniae DNA in clinical samples. J Med Microbiol
53: 189-195
[Abstract] [Full Text] -
McIntosh, E D G
(2003). How many episodes of hospital care might be prevented by widespread uptake of pneumococcal conjugate vaccine?. Arch. Dis. Child.
88: 859-861
[Abstract] [Full Text] -
Verhelst, R., Kaijalainen, T., De Baere, T., Verschraegen, G., Claeys, G., Van Simaey, L., De Ganck, C., Vaneechoutte, M.
(2003). Comparison of Five Genotypic Techniques for Identification of Optochin-Resistant Pneumococcus-Like Isolates. J. Clin. Microbiol.
41: 3521-3525
[Abstract] [Full Text] -
Scott, J. A. G., Marston, E. L., Hall, A. J., Marsh, K.
(2003). Diagnosis of Pneumococcal Pneumonia by psaA PCR Analysis of Lung Aspirates from Adult Patients in Kenya. J. Clin. Microbiol.
41: 2554-2559
[Abstract] [Full Text] -
Facklam, R.
(2002). What Happened to the Streptococci: Overview of Taxonomic and Nomenclature Changes. Clin. Microbiol. Rev.
15: 613-630
[Abstract] [Full Text] -
McIntosh, E D G, Booy, R
(2002). Invasive pneumococcal disease in England and Wales: what is the true burden and what is the potential for prevention using 7 valent pneumococcal conjugate vaccine?. Arch. Dis. Child.
86: 403-406
[Abstract] [Full Text] -
Virkki, R, Juven, T, Rikalainen, H, Svedstrom, E, Mertsola, J, Ruuskanen, O
(2002). Differentiation of bacterial and viral pneumonia in children. Thorax
57: 438-441
[Abstract] [Full Text] -
McIntosh, K.
(2002). Community-Acquired Pneumonia in Children. NEJM
346: 429-437
[Full Text] -
McAvin, J. C., Reilly, P. A., Roudabush, R. M., Barnes, W. J., Salmen, A., Jackson, G. W., Beninga, K. K., Astorga, A., McCleskey, F. K., Huff, W. B., Niemeyer, D., Lohman, K. L.
(2001). Sensitive and Specific Method for Rapid Identification of Streptococcus pneumoniae Using Real-Time Fluorescence PCR. J. Clin. Microbiol.
39: 3446-3451
[Abstract] [Full Text] -
Falguera, M., Sacristan, O., Nogues, A., Ruiz-Gonzalez, A., Garcia, M., Manonelles, A., Rubio-Caballero, M.
(2001). Nonsevere Community-Acquired Pneumonia: Correlation Between Cause and Severity or Comorbidity. Arch Intern Med
161: 1866-1872
[Abstract] [Full Text] -
Corless, C. E., Guiver, M., Borrow, R., Edwards-Jones, V., Fox, A. J., Kaczmarski, E. B.
(2001). Simultaneous Detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in Suspected Cases of Meningitis and Septicemia Using Real-Time PCR. J. Clin. Microbiol.
39: 1553-1558
[Abstract] [Full Text] -
Morrison, K. E., Lake, D., Crook, J., Carlone, G. M., Ades, E., Facklam, R., Sampson, J. S.
(2000). Confirmation of psaA in All 90 Serotypes of Streptococcus pneumoniae by PCR and Potential of This Assay for Identification and Diagnosis. J. Clin. Microbiol.
38: 434-437
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»