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Community-acquired pneumonia in children due to Mycoplasma pneumoniae: Diagnostic performance of a seminested 16S rDNA-PCR
Diagnostic Microbiology and Infectious Disease 39 (2001) 15–19
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Community-acquired pneumonia in children due to Mycoplasma pneumoniae: Diagnostic performance of a seminested 16S rDNA-PCR David Nadala,*, Walter Bossartb, Franziska Zucola, Felicitas Steinera, Christoph Bergera, Ulrich Lipsc, Martin Altweggd a
Division of Infectious Diseases, University Children’s Hospital of Zurich, CH-8032 Zurich, Switzerland b Institute of Medical Virology, University of Zurich, CH-8028 Zurich, Switzerland c Department of Pediatrics, University Children’s Hospital of Zurich, Zurich, Switzerland d Department of Medical Microbiology, University of Zurich, CH-8028 Zurich, Switzerland
Abstract A 16S rDNA-PCR assay for Mycoplasma pneumoniae applied to nasopharyngeal secretion (NPS) or pharyngeal swab (PS) from children with community-acquired pneumonia (CAP) was prospectively compared to serological tests including complement fixation (CF) test, a -capture enzyme immuno assay (EIA) for the detection of specific IgM, and an EIA for the detection of specific IgG. During a 24-months-period diagnosis of active M. pneumoniae infection was established in 32 (12.6%) of 253 patients for whom paired sera were available. In the acute phase, the sensitivities of PCR from NPS and PS, CF test, IgM EIA, and IgG EIA were 90.0%, 79.3%, 46.9%, 78.1%, and 59.4%, respectively. The corresponding specificities were 98.1%, 98.6%, 97.6%, 87.1%, and 72.4%, respectively. Thus, the 16S rDNA-PCR assay provides a highly sensitive and accurate tool for the rapid diagnosis of M. pneumoniae infection in children with CAP. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction Community-acquired pneumonia (CAP) is common among children seen in primary care. In industrialized countries, CAP occurs in children ⱕ5 years of age with an incidence of 7– 40 cases/1000 children/year and in children ⬎5 years of age with an incidence of 16.2 cases/1000 children/year (Hammerschlag, 1995; Heiskanen-Kosma et al., 1998; Jokinen et al., 1993). The etiologic agents include respiratory viruses and bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Chlamydia pneumoniae and Mycoplasma pneumoniae. Unfortunately, neither clinical nor radiological signs and symptoms allow for an etiological classification. Whereas viral CAP can be established in the majority of cases by rapid detection of the causative agent in nasopharyngeal secretions (NPS) by either antigen detection tests or PCR, the diagnosis of bacterial pneumonia requires either detection of the microorganism by culture from blood, lung aspirates or pleural secretions, or the detection of specific antibodies in serum specimens. Thus, the diagnosis of bacterial CAP re-
quires invasive procedures which are not routinely performed in pediatric practices. Moreover, specific antibody responses may still be undetectable in the acute phase of the disease (Waris et al., 1998). Therefore, antimicrobial therapeutic management of CAP presents a challenge in clinical practice. The availability of accurate diagnostic tools to rapidly recognize bacterial agents is a prerequisite for the timely initiation of adequate antimicrobial treatment in order to avoid unnecessary exposure of patients and their endogenous bacterial flora to antibiotics and, thus, to minimize the emergence of resistant bacteria. This prospective study in children with CAP was aimed at evaluating the diagnostic value of PCR for M. pneumoniae applied to NPS and pharyngeal swabs (PS) in comparison to serological tests including complement fixation (CF) test and enzyme immunoassay (EIA).
2. Materials and methods 2.1. Patients and specimens
* Corresponding author. Tel.: ⫹ 41 1 266 7111; fax: ⫹ 41 1 266 7157. E-mail address: [email protected] (D. Nadal).
All patient samples were collected during a prospective, single-center study on CAP conducted at the University
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D. Nadal et al. / Diagnostic Microbiology and Infectious Disease 39 (2001) 15–19
Children’s Hospital of Zurich between November 1, 1997 and October 31, 1999. Inclusion criteria for the patients were ages of 1 month to 16 years, an initial visit to the emergency room, radiologically documented CAP, and informed consent by the patients and/or their caregivers. Patients with hospital-acquired pneumonia, known immunodeficiencies, or chronic lung disorders were excluded. NPS were obtained at visit 1 (day 0), PS at visits 1, 2 (day 10 –14), and 3 (day 24 –28), and blood specimens at visits 1 and 3. NPS were collected with a disposable mucus extractor (Vigon, Ecouen, France) and stored at 4°C. Cottontipped PS were used to recover material from the space between the palatine arches and placed immediately in transport medium (BBL Port-A-Cul™, Becton Dickinson, Cockeysville, MD). Both NPS and PS were sent to the Department of Medical Microbiology, Zurich within 24 hours for PCR analysis. Sera were frozen at ⫺70°C until analysis. The study was approved by the institutional ethics committee.
acute-phase and the convalescent-phase serum specimen, or 6) a specific IgG titer ⱖ10 U in the acute-phase specimen. The interpretative criteria for the EIAs are consistent with the recommendations of the manufacturer as outlined on the package insert.
2.2. Serology for M. pneumoniae
2.5. Positive and negative controls
Antibodies against M. pneumoniae were determined using standard CF test or EIA technology. Acute and convalescent-phase serum specimens from each patient were tested for complement fixation, IgM, and IgG antibodies in the same run, respectively. Materials for the CF test were purchased from different sources (antigens, complement and control sera from Virion/MicrobeScope, Ru¨schlikon, Switzerland; sheep erythrocytes from bioMe´rieux, Marcy l’Etoile, France; anti-sheep erythrocyte antibodies from Dade Behring, Liederbach, Germany); the CF test was performed according to standard techniques (Leland, 1992). EIAs were performed using commercial systems (Platelia威 M. pneumoniae IgG/IgM, Sanofi Pasteur Diagnostics, Marnes la Coquette, France) for IgG and IgM antibody detection, the IgG system being a conventional indirect EIA, the IgM system an immunocapture (-capture) assay. All reagents and controls needed were included in the kits. The assays were performed according to the instructions of the manufacturer.
One ampoule of lyophilized M. pneumoniae antigen (strain ATCC 15531) prepared to be used in the complement fixation test (Institute VIRION Ltd., Ru¨schlikon, Switzerland) was resuspended in 1 ml of 0.85% NaCl solution, diluted 10⫺2 and centrifuged at 14,000 g for 10 min at room temperature. Most of the supernatant was discarded. The remaining 20 l including pellet were dried for 1 h in a vacuum centrifuge. The dried pellet was stored at 4°C until DNA extraction was performed as described above for PS and NPS pellets. As a negative control, Escherichia coli (strain MM 633) grown on blood agar was suspended in 200 ml 0.85% NaCl solution to a concentration equivalent to McFarland standard 1. The cell suspension was aliquoted into 0.5 ml portions, centrifuged at 14,000 g for 10 min at room temperature. After discarding 480 l supernatant the remaining 20 l were dried, stored and extracted as described above.
2.3. Diagnosis of active M. pneumoniae infection
PCR was performed according to Tjhie et al. 1994, with some modifications. For amplification, primers Mpne-1 and Mpne-2 (corresponding to M. pneumoniae 1 and 2) were used. The hybridization probe pMpne (originally named Probe GPO-1) was used for seminested reamplification in combination with primer Mpne-2. In all our amplifications, contaminations due to product carry-over from previous amplifications were prevented by using dUTP instead of dTTP and by the addition of uracil-DNA-glycosylase (UNG; Epicentre Technologies, Madison, WI). Amplifications were done in a final volume of 50 l containing 200 M of each deoxynucleotide, 2.5 units of AmpliTaq Gold polymerase with the appropriate amount of its optimized buffer (Perkin-Elmer, Norwalk, USA), 25 pmol of each
The diagnosis of active infection with M. pneumoniae was based solely on serological results. The criteria for establishing the serodiagnosis of active infection with M. pneumoniae were: 1) seroconversion detected by CF test or by IgG EIA (for IgG an increase from ⬍10 U in the acute-phase serum specimen to ⬎20 U for a convalescentphase serum specimen was considered as seroconversion), 2) a reciprocal titer ⱖ80 in the CF test in the acute-phase and the convalescent-phase serum specimen, 3) a fourfold titer rise in the CF test in paired sera, 4) presence of specific IgM in both the acute-phase and the convalescent-phase serum specimen, 5) a specific IgG titer ⱖ40 U in the
2.4. DNA extraction PS were thoroughly washed in 2 ml 0.85% NaCl. These suspensions and the NPS were then centrifuged at 14,000 g for 10 min at room temperature. The resulting pellets were suspended in 200 l digestion buffer (50 mM Tris-HCl, pH 8.5, 1 mM EDTA, 0.5% SDS, 200 g/ml proteinase K) and incubated at 55°C for 90 min with agitation. DNA was extracted using QIAamp™ DNA binding columns (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol, except for the final step using only 100 l (instead of 200 l) of elution buffer AE to increase the concentration of DNA for subsequent PCR analysis.
2.6. 16S rDNA-PCR for M. pneumoniae
D. Nadal et al. / Diagnostic Microbiology and Infectious Disease 39 (2001) 15–19
primer, 0.5 U UNG, and 5 l of DNA extract. Each PCR run included 5 l DNA extract of positive and negative controls. All PCRs were performed on a GeneAmp™ PCR System 9600 (Perkin-Elmer) and included an initial activation step for the polymerase at 95°C for 12 min. Amplifications with primer pairs Mpne-1/Mpne-2 were done for 40 cycles of 1 min at 94°C for denaturation, 1 min at 60°C for annealing, and 1 min at 72°C for extension. For seminested reamplification with primers pMpne/Mpne-2, 5 l amplicon were used as template and amplified for 15 cycles using the same conditions as for primary amplification, except that UNG was omitted. PCR products (10 l) were separated by electrophoresis on a 2% (w/v) agarose gel, stained with ethidium bromide, and detected under UV light. 2.7. Statistics The ages of the patients seronegative and seropositive for M. pneumoniae were compared using the Mann-U-Whitney test. P values ⬍ 0.05 were regarded as significant.
3. Results During the 24-month period 490 children were seen at the emergency room and diagnosed as having CAP. After obtaining informed consent, 356 (72.7%) of these children could be enrolled in the study. Thirty-three (9.3%) of the 356 children were later excluded because reevaluation of chest X-rays did not confirm the presence of pneumonia and 70 (19.7%) children because no paired sera had been obtained. Serodiagnosis of active M. pneumoniae infection was established in 32 (12.6%) children and disproven in the other 221 (87.4%) children of the 253 children remaining in the study. Polymerase chain reaction was positive from NPS in 27 out of 30 patients and from PS in 23 out of 29 patients with serologically proven acute infection with M. pneumoniae as well as from NPS in 4 out of 211 patients and from PS in 3 out of 215 patients with serologically disproven acute infection with this agent. Sensitivities and specificities as well as positive and negative predictive values of PCR and serological tests for the acute phase (visit 1) are summarized in Table 1. Combination of the results of PCR from NPS and PS, respectively, from the acute phase increased the sensitivity of PCR to 92.6% and decreased the specificity of PCR to 97.7%. Among patients with serologically proven M. pneumoniae infection, PCR for M. pneumoniae was still positive from PS in 20 (76.9%) out of 26 patients tested at visit 2 and from PS in 17 (56.7%) of the 30 patients tested in the convalescent phase (visit 3). In patients with CAP serologically not positive for M. pneumoniae, PCR was positive from PS in 2 (1.1%) of 186 patients tested on visit 2 and from PS in 3 (1.4%) of 217 patients tested on visit 3. Only one of the patients with no active M. pneumoniae infection
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Table 1 Performance of the tests employed for the detection of active M. pneumoniae infection at visit 1 in relation to the serological results on paired sera serving as gold standard Test
Sensitivity (%) Specificity (%) PPVa (%) NPVb (%)
PCR from NPS 90.0 PCR from PS 79.3 CF test 46.9 IgM EIA 78.1 IgG EIA 59.4 a b
98.1 98.6 97.6 87.1 72.4
87.1 88.5 75.0 67.6 41.4
98.6 95.9 92.2 96.3 92.1
PPV, positive predictive value. NPV, negative predictive value.
as defined by serology on paired sera showed positive PCRs on more than one visit. The patient was 4.5 years old, had a persistent CF test titer 1:20, neither specific IgM, nor IgG in acute and convalescent sera, and tested positive by PCR from NPS and PS at visit 1 and also from PS at visits 2 and 3. The median age of the patients with CAP due to M. pneumoniae was 7.1 years (range, 0.2–14.8) and of those with CAP not due to this bacterium was 2.5 years (0.1–14.9; P ⬍ 0.0001). Eleven (34.4%) of the 32 patients with CAP due to M. pneumoniae were ⱕ5 years of age. M. pneumoniae accounted for 6% of CAP cases in patients ⱕ5 years of age, but for 29% in older patients. There was no clear-cut seasonal clustering of CAP cases due to M. pneumoniae. Three patients with CAP due to M. pneumoniae received no antimicrobial treatment at all, 15 a -lactam antibiotic, three a -lactam antibiotic plus a macrolide, ten a macrolide, and one patient tetracycline. Thus, 18 of 32 patients were not prescribed a treatment considered effective, at least in vitro, against M. pneumoniae. Among these, follow-up PS were still PCR positive in 12 (80%) of 15 and 9 (56.3%) of 16 patients tested at visits 2 and 3, respectively. In patients receiving antimicrobial treatment considered effective against M. pneumoniae this agent was detectable by PCR from PS from the intermediate phase and from the convalescent phase in 9 (75%) of 12 and in 8 (57.1%) of 14 patients tested, respectively. The clinical outcomes of the 18 patients not prescribed treatment considered effective against M. pneumoniae did not differ from the outcomes of the 14 patients prescribed antimicrobial treatment considered effective. All patients recovered without sequelae.
4. Discussion Our prospective study reveals that in the acute phase of CAP due to M. pneumoniae the sensitivity of 16S rDNAPCR from NPS is superior to that of serological tests and of PCR from PS (Table 1). Further, the specificities of 16S rDNA-PCR from NPS and PS were similar to that of the CF test and superior to those of specific IgM or specific IgG (Table 1).
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The standard laboratory methods for the diagnosis of M. pneumoniae as etiologic agent for CAP have been culture and serology (Block et al., 1995; Dorigo-Zetsma et al., 1999; Marmion et al., 1993; Waris et al., 1998). In the present study we did not perform cultures for M. pneumoniae because of the logistical problems associated with the rapid transport of specimens to the laboratory necessary to obtain reliable culture results. However, since only patients with paired sera and without known or overt immunodeficiency were included in the evaluation, the criteria used to judge for the presence of acute infection with M. pneumoniae including seroconversion, significant increases of specific antibody titers in the CF test or of specific IgG, or the persistence of IgM can be regarded as gold standard. Notably, our rate of CAP due to M. pneumoniae was similar to the rates in most of the recent studies on CAP in children (Dorigo-Zetsma et al., 1999; Ieven et al., 1996; Waris et al., 1998; Wubbel et al., 1999) or in adults (Layani-Milon et al., 1999). However, rates of CAP due to M. pneumoniae in children and adults as high as 15% to 29.5% (Block et al., 1995; Foy, 1993; Harris et al., 1998; Heiskanen-Kosma et al., 1998) and as low as 1% to 3% have been communicated (Gro¨ndahl et al., 1999; Vuori et al., 1998). These differences in relative frequency of CAP due to M. pneumoniae may be attributed to the presence or absence of community outbreaks during the study periods. Epidemics of M. pneumoniae appear to occur at intervals of 4 –7 years (Foy, 1993). As noted in epidemic free intervals (Foy, 1993) we did not observe a seasonal clustering of CAP cases due to M. pneumoniae in the late summer and early fall. Another potential reason for an overall frequency of CAP due to M. pneumoniae ⱖ25% is that some authors assumed acute infection with this agent not only in cases with serological evidence and/or positive cultures but also in cases positive by PCR only, without considering a possibly low specificity of this assay (Block et al., 1995; Harris et al., 1998). In the acute phase of CAP, IgM and IgG EIA serological methods missed diagnosis of active infection with M. pneumoniae in more than one to two of every five children with serologically confirmed infection with this agent. Whereas the sensitivity of 78.1% of the commercial IgM-capture EIA used in our study is in agreement with the reported sensitivity of 79% of the same assay in children with CAP, the sensitivity of the IgG EIA was almost one fifth lower than the one found in the same report (59.4% vs. 78%) (Waris et al., 1998). The reason for this discrepancy remains unknown. The diagnostic performance of the CF test in the acute-phase was even worse missing more than half of the infections due to M. pneumoniae. This confirms the extremely poor value of the CF test as a diagnostic tool for CAP due to M. pneumoniae at the initial visit. Nevertheless, the CF test may serve for confirmatory purposes when convalescent-phase sera are available. In this situation an overall sensitivity of ⬎90% can be expected (Jacobs, 1993). In our study the CF test was used as an additional standard
to assess the presence of an acute infection due to M. pneumoniae. The finding of greatest interest for clinical practice in our study was the higher sensitivity and specificity of the 16S rDNA-PCR applied to NPS or PS for the detection of M. pneumoniae in the acute phase of CAP as compared to serology. Moreover, the positive predictive values of the 16S rDNA-PCR on NPS and PS considerably exceeded that of the CF test, the specific IgM, or specific IgG EIA (Table 1). The negative predictive values of the 16S rDNA-PCR on NPS and PS were similar to those of the CF test, specific IgM, and specific IgG EIA (Table 1). The utility and value of PCR techniques in diagnosing M. pneumoniae respiratory tract infections has also been reported by several groups (Abele-Horn et al., 1998; Ieven et al., 1996; Tjhie et al., 1994). Another interesting observation was the detection of M. pneumoniae by the 16S rDNA-PCR in PS from the convalescent-phase in more than half of the patients independent of whether treatment, effective at least in vitro, against this organism had been prescribed or not. This contrasts the findings of studies showing eradication of M. pneumoniae from the pharynx in ⬎86% following treatment with macrolides to which this bacterium is susceptible (Block et al., 1995; DorigoZetsma et al., 1999; Harris et al., 1998; Wubbel et al., 1999). However, in these studies eradication was assessed either by culture which in independent studies showed a sensitivity of 61.5% compared to 16S rRNA-PCR (Ieven et al., 1996) or of 64% compared to serodiagnosis (Jacobs, 1993), or by a PCR test for which sensitivity was not provided (Block et al., 1995; Harris et al., 1998; Wubbel et al., 1999) or as low as 78% (Dorigo-Zetsma et al., 1999). Since we required clear serologic evidence for the diagnosis of acute infection with M. pneumoniae, it seems unlikely that a preexisting carrier state (Foy, 1993) accounted for our results. A possible reason for the prolonged detection of M. pneumoniae by the 16S rDNA-PCR, even following effective antimicrobial treatment, could have been the detection of persisting bacterial DNA of dead microorganisms. Nevertheless, in our cohort of patients with CAP due to M. pneumoniae no sequelae were observed, regardless of whether effective treatment was prescribed or not. Whether diagnosis of CAP due to M. pneumoniae is important to patient outcome needs to be determined in studies enrolling larger cohorts of children and addressing specifically this question. Finally, positive PCR results in CAP patients with serologically disproven acute infection with M. pneumoniae could represent either false positive PCR results (carry-over contamination), the detection of a carrier state not manifesting systemic antibody responses, or false negative serology despite active infection.
Acknowledgment The authors wish to express their gratitude to the medical and the nursing staff of the emergency room of the Univer-
D. Nadal et al. / Diagnostic Microbiology and Infectious Disease 39 (2001) 15–19
sity Children’s Hospital of Zurich for recruitment of patients and specimen collection in the acute-phase, the physicians in private practice for their cooperation allowing for follow-up investigations, and the patients and their parents for their participation in the study. Furthermore, the superb laboratory work of Pia Beck and Inge Perschil is acknowledged as well as Alexander von Graevenitz for critically reading the manuscript. The study was in part financially supported by a grant from Pfizer AG, Zurich, Switzerland.
References Abele-Horn, M., Busch, U., Nitschko, H., Jacobs, E., Bax, R., Pfaff, F., Schaffer, B., & Heesemann, J. (1998). Molecular approaches to diagnosis of pulmonary diseases due to Mycoplasma pneumoniae. J Clin Microbiol 36, 548 –551. Block, S., Hedrick, J., Hammerschlag, M. R., Casell, G. H., & Craft, J. C. (1995). Mycoplasma pneumoniae and Chlamydia pneumoniae in pediatric community-acquired pneumonia: comparative efficacy and safety of clarithromycin vs. erythromycin ethylsuccinate. Pediatr Infect Dis J 14, 471– 477. Dorigo-Zetsma, J. W., Zaat, S. A. J., Wertheim-van Dillen, P. M. E., Spanjaard, L., Rijntes, J., van Waveren, G., Jensen, J. S., Angulo, A. F., & Dankert, J. (1999). Comparison of PCR, culture, and serological tests for diagnosis of Mycoplasma pneumoniae respiratory tract in children. J Clin Microbiol 37, 14 –17. Foy, H. M. (1993). Infections caused by Mycoplasma pneumoniae and possible carrier state in different populations of patients. Clin Infect Dis 17 (Suppl 1):S37–S46. Gro¨ndahl, B., Puppe, W., Hoppe, A., Ku¨hne, I., Weigl, J. A. I., & Schmitt, H-J. (1999). Rapid identification of nine microorganisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-PCR: feasibility study. J Clin Microbiol 37, 1–7. Hammerschlag, M. R. (1995). Atypical pneumonias in children. Adv Pediatric Infect Dis 10, 1–39. Harris, J-A. H., Kolokathis, A., Campbell, M., Casell, G. H., & Hammerschlag, M. R. (1998). Safety and efficacy of azithromycin in the treatment of community-acquired pneumonia in children. Pediatr Infect Dis J 17, 865– 871. Heiskanen-Kosma, T., Korppi, M., Jokinen, C., Kurki, S., Heiskanen, L., Juvonen, H., Kallinen, S., Ste´n, M., Tarkainen, A., Pirjo-Riita, R., Kleemola, M., Meka¨la¨, P. H., & Leinone, M. (1998). Etiology of
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childhood pneumonia: serologic results of a prospective, populationbased study. Pediatr Infect Dis J 17, 986 –991. Ieven, M., Ursi, D., Van Bever, H., Quint, W., Niesters, H. G. M., & Goossens, H. (1996). Detection of Mycoplasma pneumoniae by two polymerase chain reactions and role of M. pneumoniae in acute respiratory tract infections in pediatric patients. J Infect Dis 173, 1445– 1452. Jacobs, E. (1993). Serological diagnosis of Mycoplasma pneumoniae infections: a critical review of current procedures. Clin Infect Dis 17 (Suppl 1):S79 –S82. Jokinen, C., Heiskanen, L., Juvonen, H., Kallinen, S., Karkola, K., Korppi, M., Kurki, S., Ro¨nnberg, P-R., Seppa¨, A., Soimakallio, S., Ste´n, M., Tanska, S., Tarkainen, A., Tukiainen, H., Pyo¨ra¨la¨, K., & Ma¨kala¨, P. H. (1993). Incidence of community-acquired pneumonia in the population of four municipalities in eastern Finland. Am J Epidemiol 137, 977– 988. Layani-Milon, M-P., Gras, I., Valette, M., Luciani, J., Stagnara, J., Aymard, M., & Lina, B. (1999). Incidence of upper respiratory tract Mycoplasma pneumoniae infections among outpatients in RhoˆneAlpes, France, during five successive winter periods. J Clin Microbiol 36, 1721–1726. Leland, D. S. (1992). Concepts in clinical diagnostic virology. In: E. H. Lennette (Ed), Laboratory diagnosis of viral infection, Marcel Dekker, New York, Basel, Hong Kong, pp. 37–38. Marmion, B. P., Williamson, J., Worswick, D. A., Kok, T-W., & Harris, R. J. (1993). Experience with newer techniques for the laboratory detection of Mycoplasma pneumoniae infection: Adelaide, 1978 –1992. Clin Infect Dis 17 (Suppl 1):S90 –S99. Tjhie, J. H. T., van Kuppeveld, F. J. M., Roosendaal, R., Melchers, W. J. G., Gordijn, R., MacLaren, D. M., Walboomers, J. M. M., Meijer, C. J. L. M., & van der Brule, A. J. C. (1994). Direct PCR enables detection of Mycoplasma pneumoniae in patients with respiratory tract infections. J Clin Microbiol 32, 11–16. Vuori, E., Peltola, H., Kallio, M. J. T., Leinonen, M., Hedman, K., & Group, S-TS. (1998). Etiology of pneumonia and other common childhood infections requiring hospitalization and parenteral antimicrobial therapy. Clin Infect Dis 27, 566 –572. Waris, M. E., Toikka, P., Saarinen, T., Nikkari, S., Meurman, O., Vainionpa¨a¨, R., Mertsola, J., & Ruuskanen, O. (1998). Diagnosis of Mycoplasma pneumoniae pneumonia in children. J Clin Microbiol 36, 3155– 3159. Wubbel, L., Muniz, L., Ahmed, A., Trujillo, M., Carubelli, C., McCoig, C., Abramo, T., Leinonen, M., & McCracken, G. H. (1999). Etiology and treatment of community-acquired pneumonia in ambulatory pediatrics. Pediatr Infect Dis J 18, 98 –104.
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Community-acquired pneumonia in children due to Mycoplasma pneumoniae: Diagnostic performance of a seminested 16S rDNA-PCR David Nadala,*, Walter Bossartb, Franziska Zucola, Felicitas Steinera, Christoph Bergera, Ulrich Lipsc, Martin Altweggd a
Division of Infectious Diseases, University Children’s Hospital of Zurich, CH-8032 Zurich, Switzerland b Institute of Medical Virology, University of Zurich, CH-8028 Zurich, Switzerland c Department of Pediatrics, University Children’s Hospital of Zurich, Zurich, Switzerland d Department of Medical Microbiology, University of Zurich, CH-8028 Zurich, Switzerland
Abstract A 16S rDNA-PCR assay for Mycoplasma pneumoniae applied to nasopharyngeal secretion (NPS) or pharyngeal swab (PS) from children with community-acquired pneumonia (CAP) was prospectively compared to serological tests including complement fixation (CF) test, a -capture enzyme immuno assay (EIA) for the detection of specific IgM, and an EIA for the detection of specific IgG. During a 24-months-period diagnosis of active M. pneumoniae infection was established in 32 (12.6%) of 253 patients for whom paired sera were available. In the acute phase, the sensitivities of PCR from NPS and PS, CF test, IgM EIA, and IgG EIA were 90.0%, 79.3%, 46.9%, 78.1%, and 59.4%, respectively. The corresponding specificities were 98.1%, 98.6%, 97.6%, 87.1%, and 72.4%, respectively. Thus, the 16S rDNA-PCR assay provides a highly sensitive and accurate tool for the rapid diagnosis of M. pneumoniae infection in children with CAP. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction Community-acquired pneumonia (CAP) is common among children seen in primary care. In industrialized countries, CAP occurs in children ⱕ5 years of age with an incidence of 7– 40 cases/1000 children/year and in children ⬎5 years of age with an incidence of 16.2 cases/1000 children/year (Hammerschlag, 1995; Heiskanen-Kosma et al., 1998; Jokinen et al., 1993). The etiologic agents include respiratory viruses and bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Chlamydia pneumoniae and Mycoplasma pneumoniae. Unfortunately, neither clinical nor radiological signs and symptoms allow for an etiological classification. Whereas viral CAP can be established in the majority of cases by rapid detection of the causative agent in nasopharyngeal secretions (NPS) by either antigen detection tests or PCR, the diagnosis of bacterial pneumonia requires either detection of the microorganism by culture from blood, lung aspirates or pleural secretions, or the detection of specific antibodies in serum specimens. Thus, the diagnosis of bacterial CAP re-
quires invasive procedures which are not routinely performed in pediatric practices. Moreover, specific antibody responses may still be undetectable in the acute phase of the disease (Waris et al., 1998). Therefore, antimicrobial therapeutic management of CAP presents a challenge in clinical practice. The availability of accurate diagnostic tools to rapidly recognize bacterial agents is a prerequisite for the timely initiation of adequate antimicrobial treatment in order to avoid unnecessary exposure of patients and their endogenous bacterial flora to antibiotics and, thus, to minimize the emergence of resistant bacteria. This prospective study in children with CAP was aimed at evaluating the diagnostic value of PCR for M. pneumoniae applied to NPS and pharyngeal swabs (PS) in comparison to serological tests including complement fixation (CF) test and enzyme immunoassay (EIA).
2. Materials and methods 2.1. Patients and specimens
* Corresponding author. Tel.: ⫹ 41 1 266 7111; fax: ⫹ 41 1 266 7157. E-mail address: [email protected] (D. Nadal).
All patient samples were collected during a prospective, single-center study on CAP conducted at the University
0732-8893/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 2 - 8 8 9 3 ( 0 0 ) 0 0 2 1 6 - 9
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D. Nadal et al. / Diagnostic Microbiology and Infectious Disease 39 (2001) 15–19
Children’s Hospital of Zurich between November 1, 1997 and October 31, 1999. Inclusion criteria for the patients were ages of 1 month to 16 years, an initial visit to the emergency room, radiologically documented CAP, and informed consent by the patients and/or their caregivers. Patients with hospital-acquired pneumonia, known immunodeficiencies, or chronic lung disorders were excluded. NPS were obtained at visit 1 (day 0), PS at visits 1, 2 (day 10 –14), and 3 (day 24 –28), and blood specimens at visits 1 and 3. NPS were collected with a disposable mucus extractor (Vigon, Ecouen, France) and stored at 4°C. Cottontipped PS were used to recover material from the space between the palatine arches and placed immediately in transport medium (BBL Port-A-Cul™, Becton Dickinson, Cockeysville, MD). Both NPS and PS were sent to the Department of Medical Microbiology, Zurich within 24 hours for PCR analysis. Sera were frozen at ⫺70°C until analysis. The study was approved by the institutional ethics committee.
acute-phase and the convalescent-phase serum specimen, or 6) a specific IgG titer ⱖ10 U in the acute-phase specimen. The interpretative criteria for the EIAs are consistent with the recommendations of the manufacturer as outlined on the package insert.
2.2. Serology for M. pneumoniae
2.5. Positive and negative controls
Antibodies against M. pneumoniae were determined using standard CF test or EIA technology. Acute and convalescent-phase serum specimens from each patient were tested for complement fixation, IgM, and IgG antibodies in the same run, respectively. Materials for the CF test were purchased from different sources (antigens, complement and control sera from Virion/MicrobeScope, Ru¨schlikon, Switzerland; sheep erythrocytes from bioMe´rieux, Marcy l’Etoile, France; anti-sheep erythrocyte antibodies from Dade Behring, Liederbach, Germany); the CF test was performed according to standard techniques (Leland, 1992). EIAs were performed using commercial systems (Platelia威 M. pneumoniae IgG/IgM, Sanofi Pasteur Diagnostics, Marnes la Coquette, France) for IgG and IgM antibody detection, the IgG system being a conventional indirect EIA, the IgM system an immunocapture (-capture) assay. All reagents and controls needed were included in the kits. The assays were performed according to the instructions of the manufacturer.
One ampoule of lyophilized M. pneumoniae antigen (strain ATCC 15531) prepared to be used in the complement fixation test (Institute VIRION Ltd., Ru¨schlikon, Switzerland) was resuspended in 1 ml of 0.85% NaCl solution, diluted 10⫺2 and centrifuged at 14,000 g for 10 min at room temperature. Most of the supernatant was discarded. The remaining 20 l including pellet were dried for 1 h in a vacuum centrifuge. The dried pellet was stored at 4°C until DNA extraction was performed as described above for PS and NPS pellets. As a negative control, Escherichia coli (strain MM 633) grown on blood agar was suspended in 200 ml 0.85% NaCl solution to a concentration equivalent to McFarland standard 1. The cell suspension was aliquoted into 0.5 ml portions, centrifuged at 14,000 g for 10 min at room temperature. After discarding 480 l supernatant the remaining 20 l were dried, stored and extracted as described above.
2.3. Diagnosis of active M. pneumoniae infection
PCR was performed according to Tjhie et al. 1994, with some modifications. For amplification, primers Mpne-1 and Mpne-2 (corresponding to M. pneumoniae 1 and 2) were used. The hybridization probe pMpne (originally named Probe GPO-1) was used for seminested reamplification in combination with primer Mpne-2. In all our amplifications, contaminations due to product carry-over from previous amplifications were prevented by using dUTP instead of dTTP and by the addition of uracil-DNA-glycosylase (UNG; Epicentre Technologies, Madison, WI). Amplifications were done in a final volume of 50 l containing 200 M of each deoxynucleotide, 2.5 units of AmpliTaq Gold polymerase with the appropriate amount of its optimized buffer (Perkin-Elmer, Norwalk, USA), 25 pmol of each
The diagnosis of active infection with M. pneumoniae was based solely on serological results. The criteria for establishing the serodiagnosis of active infection with M. pneumoniae were: 1) seroconversion detected by CF test or by IgG EIA (for IgG an increase from ⬍10 U in the acute-phase serum specimen to ⬎20 U for a convalescentphase serum specimen was considered as seroconversion), 2) a reciprocal titer ⱖ80 in the CF test in the acute-phase and the convalescent-phase serum specimen, 3) a fourfold titer rise in the CF test in paired sera, 4) presence of specific IgM in both the acute-phase and the convalescent-phase serum specimen, 5) a specific IgG titer ⱖ40 U in the
2.4. DNA extraction PS were thoroughly washed in 2 ml 0.85% NaCl. These suspensions and the NPS were then centrifuged at 14,000 g for 10 min at room temperature. The resulting pellets were suspended in 200 l digestion buffer (50 mM Tris-HCl, pH 8.5, 1 mM EDTA, 0.5% SDS, 200 g/ml proteinase K) and incubated at 55°C for 90 min with agitation. DNA was extracted using QIAamp™ DNA binding columns (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol, except for the final step using only 100 l (instead of 200 l) of elution buffer AE to increase the concentration of DNA for subsequent PCR analysis.
2.6. 16S rDNA-PCR for M. pneumoniae
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primer, 0.5 U UNG, and 5 l of DNA extract. Each PCR run included 5 l DNA extract of positive and negative controls. All PCRs were performed on a GeneAmp™ PCR System 9600 (Perkin-Elmer) and included an initial activation step for the polymerase at 95°C for 12 min. Amplifications with primer pairs Mpne-1/Mpne-2 were done for 40 cycles of 1 min at 94°C for denaturation, 1 min at 60°C for annealing, and 1 min at 72°C for extension. For seminested reamplification with primers pMpne/Mpne-2, 5 l amplicon were used as template and amplified for 15 cycles using the same conditions as for primary amplification, except that UNG was omitted. PCR products (10 l) were separated by electrophoresis on a 2% (w/v) agarose gel, stained with ethidium bromide, and detected under UV light. 2.7. Statistics The ages of the patients seronegative and seropositive for M. pneumoniae were compared using the Mann-U-Whitney test. P values ⬍ 0.05 were regarded as significant.
3. Results During the 24-month period 490 children were seen at the emergency room and diagnosed as having CAP. After obtaining informed consent, 356 (72.7%) of these children could be enrolled in the study. Thirty-three (9.3%) of the 356 children were later excluded because reevaluation of chest X-rays did not confirm the presence of pneumonia and 70 (19.7%) children because no paired sera had been obtained. Serodiagnosis of active M. pneumoniae infection was established in 32 (12.6%) children and disproven in the other 221 (87.4%) children of the 253 children remaining in the study. Polymerase chain reaction was positive from NPS in 27 out of 30 patients and from PS in 23 out of 29 patients with serologically proven acute infection with M. pneumoniae as well as from NPS in 4 out of 211 patients and from PS in 3 out of 215 patients with serologically disproven acute infection with this agent. Sensitivities and specificities as well as positive and negative predictive values of PCR and serological tests for the acute phase (visit 1) are summarized in Table 1. Combination of the results of PCR from NPS and PS, respectively, from the acute phase increased the sensitivity of PCR to 92.6% and decreased the specificity of PCR to 97.7%. Among patients with serologically proven M. pneumoniae infection, PCR for M. pneumoniae was still positive from PS in 20 (76.9%) out of 26 patients tested at visit 2 and from PS in 17 (56.7%) of the 30 patients tested in the convalescent phase (visit 3). In patients with CAP serologically not positive for M. pneumoniae, PCR was positive from PS in 2 (1.1%) of 186 patients tested on visit 2 and from PS in 3 (1.4%) of 217 patients tested on visit 3. Only one of the patients with no active M. pneumoniae infection
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Table 1 Performance of the tests employed for the detection of active M. pneumoniae infection at visit 1 in relation to the serological results on paired sera serving as gold standard Test
Sensitivity (%) Specificity (%) PPVa (%) NPVb (%)
PCR from NPS 90.0 PCR from PS 79.3 CF test 46.9 IgM EIA 78.1 IgG EIA 59.4 a b
98.1 98.6 97.6 87.1 72.4
87.1 88.5 75.0 67.6 41.4
98.6 95.9 92.2 96.3 92.1
PPV, positive predictive value. NPV, negative predictive value.
as defined by serology on paired sera showed positive PCRs on more than one visit. The patient was 4.5 years old, had a persistent CF test titer 1:20, neither specific IgM, nor IgG in acute and convalescent sera, and tested positive by PCR from NPS and PS at visit 1 and also from PS at visits 2 and 3. The median age of the patients with CAP due to M. pneumoniae was 7.1 years (range, 0.2–14.8) and of those with CAP not due to this bacterium was 2.5 years (0.1–14.9; P ⬍ 0.0001). Eleven (34.4%) of the 32 patients with CAP due to M. pneumoniae were ⱕ5 years of age. M. pneumoniae accounted for 6% of CAP cases in patients ⱕ5 years of age, but for 29% in older patients. There was no clear-cut seasonal clustering of CAP cases due to M. pneumoniae. Three patients with CAP due to M. pneumoniae received no antimicrobial treatment at all, 15 a -lactam antibiotic, three a -lactam antibiotic plus a macrolide, ten a macrolide, and one patient tetracycline. Thus, 18 of 32 patients were not prescribed a treatment considered effective, at least in vitro, against M. pneumoniae. Among these, follow-up PS were still PCR positive in 12 (80%) of 15 and 9 (56.3%) of 16 patients tested at visits 2 and 3, respectively. In patients receiving antimicrobial treatment considered effective against M. pneumoniae this agent was detectable by PCR from PS from the intermediate phase and from the convalescent phase in 9 (75%) of 12 and in 8 (57.1%) of 14 patients tested, respectively. The clinical outcomes of the 18 patients not prescribed treatment considered effective against M. pneumoniae did not differ from the outcomes of the 14 patients prescribed antimicrobial treatment considered effective. All patients recovered without sequelae.
4. Discussion Our prospective study reveals that in the acute phase of CAP due to M. pneumoniae the sensitivity of 16S rDNAPCR from NPS is superior to that of serological tests and of PCR from PS (Table 1). Further, the specificities of 16S rDNA-PCR from NPS and PS were similar to that of the CF test and superior to those of specific IgM or specific IgG (Table 1).
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The standard laboratory methods for the diagnosis of M. pneumoniae as etiologic agent for CAP have been culture and serology (Block et al., 1995; Dorigo-Zetsma et al., 1999; Marmion et al., 1993; Waris et al., 1998). In the present study we did not perform cultures for M. pneumoniae because of the logistical problems associated with the rapid transport of specimens to the laboratory necessary to obtain reliable culture results. However, since only patients with paired sera and without known or overt immunodeficiency were included in the evaluation, the criteria used to judge for the presence of acute infection with M. pneumoniae including seroconversion, significant increases of specific antibody titers in the CF test or of specific IgG, or the persistence of IgM can be regarded as gold standard. Notably, our rate of CAP due to M. pneumoniae was similar to the rates in most of the recent studies on CAP in children (Dorigo-Zetsma et al., 1999; Ieven et al., 1996; Waris et al., 1998; Wubbel et al., 1999) or in adults (Layani-Milon et al., 1999). However, rates of CAP due to M. pneumoniae in children and adults as high as 15% to 29.5% (Block et al., 1995; Foy, 1993; Harris et al., 1998; Heiskanen-Kosma et al., 1998) and as low as 1% to 3% have been communicated (Gro¨ndahl et al., 1999; Vuori et al., 1998). These differences in relative frequency of CAP due to M. pneumoniae may be attributed to the presence or absence of community outbreaks during the study periods. Epidemics of M. pneumoniae appear to occur at intervals of 4 –7 years (Foy, 1993). As noted in epidemic free intervals (Foy, 1993) we did not observe a seasonal clustering of CAP cases due to M. pneumoniae in the late summer and early fall. Another potential reason for an overall frequency of CAP due to M. pneumoniae ⱖ25% is that some authors assumed acute infection with this agent not only in cases with serological evidence and/or positive cultures but also in cases positive by PCR only, without considering a possibly low specificity of this assay (Block et al., 1995; Harris et al., 1998). In the acute phase of CAP, IgM and IgG EIA serological methods missed diagnosis of active infection with M. pneumoniae in more than one to two of every five children with serologically confirmed infection with this agent. Whereas the sensitivity of 78.1% of the commercial IgM-capture EIA used in our study is in agreement with the reported sensitivity of 79% of the same assay in children with CAP, the sensitivity of the IgG EIA was almost one fifth lower than the one found in the same report (59.4% vs. 78%) (Waris et al., 1998). The reason for this discrepancy remains unknown. The diagnostic performance of the CF test in the acute-phase was even worse missing more than half of the infections due to M. pneumoniae. This confirms the extremely poor value of the CF test as a diagnostic tool for CAP due to M. pneumoniae at the initial visit. Nevertheless, the CF test may serve for confirmatory purposes when convalescent-phase sera are available. In this situation an overall sensitivity of ⬎90% can be expected (Jacobs, 1993). In our study the CF test was used as an additional standard
to assess the presence of an acute infection due to M. pneumoniae. The finding of greatest interest for clinical practice in our study was the higher sensitivity and specificity of the 16S rDNA-PCR applied to NPS or PS for the detection of M. pneumoniae in the acute phase of CAP as compared to serology. Moreover, the positive predictive values of the 16S rDNA-PCR on NPS and PS considerably exceeded that of the CF test, the specific IgM, or specific IgG EIA (Table 1). The negative predictive values of the 16S rDNA-PCR on NPS and PS were similar to those of the CF test, specific IgM, and specific IgG EIA (Table 1). The utility and value of PCR techniques in diagnosing M. pneumoniae respiratory tract infections has also been reported by several groups (Abele-Horn et al., 1998; Ieven et al., 1996; Tjhie et al., 1994). Another interesting observation was the detection of M. pneumoniae by the 16S rDNA-PCR in PS from the convalescent-phase in more than half of the patients independent of whether treatment, effective at least in vitro, against this organism had been prescribed or not. This contrasts the findings of studies showing eradication of M. pneumoniae from the pharynx in ⬎86% following treatment with macrolides to which this bacterium is susceptible (Block et al., 1995; DorigoZetsma et al., 1999; Harris et al., 1998; Wubbel et al., 1999). However, in these studies eradication was assessed either by culture which in independent studies showed a sensitivity of 61.5% compared to 16S rRNA-PCR (Ieven et al., 1996) or of 64% compared to serodiagnosis (Jacobs, 1993), or by a PCR test for which sensitivity was not provided (Block et al., 1995; Harris et al., 1998; Wubbel et al., 1999) or as low as 78% (Dorigo-Zetsma et al., 1999). Since we required clear serologic evidence for the diagnosis of acute infection with M. pneumoniae, it seems unlikely that a preexisting carrier state (Foy, 1993) accounted for our results. A possible reason for the prolonged detection of M. pneumoniae by the 16S rDNA-PCR, even following effective antimicrobial treatment, could have been the detection of persisting bacterial DNA of dead microorganisms. Nevertheless, in our cohort of patients with CAP due to M. pneumoniae no sequelae were observed, regardless of whether effective treatment was prescribed or not. Whether diagnosis of CAP due to M. pneumoniae is important to patient outcome needs to be determined in studies enrolling larger cohorts of children and addressing specifically this question. Finally, positive PCR results in CAP patients with serologically disproven acute infection with M. pneumoniae could represent either false positive PCR results (carry-over contamination), the detection of a carrier state not manifesting systemic antibody responses, or false negative serology despite active infection.
Acknowledgment The authors wish to express their gratitude to the medical and the nursing staff of the emergency room of the Univer-
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sity Children’s Hospital of Zurich for recruitment of patients and specimen collection in the acute-phase, the physicians in private practice for their cooperation allowing for follow-up investigations, and the patients and their parents for their participation in the study. Furthermore, the superb laboratory work of Pia Beck and Inge Perschil is acknowledged as well as Alexander von Graevenitz for critically reading the manuscript. The study was in part financially supported by a grant from Pfizer AG, Zurich, Switzerland.
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