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J Clin Microbiol. Jan 1999; 37(1): 1–7.
PMCID: PMC84151

Rapid Identification of Nine Microorganisms Causing Acute Respiratory Tract Infections by Single-Tube Multiplex Reverse Transcription-PCR: Feasibility Study

Abstract

Acute respiratory tract infections (ARIs) are leading causes of morbidity and, in developing countries, mortality in children. A multiplex reverse transcription-PCR (RT-PCR) assay was developed to allow in one test the detection of nine different microorganisms (enterovirus, influenza A and B viruses, respiratory syncytial virus [RSV], parainfluenzaviruses type 1 and type 3, adenovirus, Mycoplasma pneumoniae, and Chlamydia pneumoniae) that do not usually colonize the respiratory tracts of humans but, if present, must be assumed to be the cause of respiratory disease. Clinical samples from 1,118 children admitted to the Department of Pediatrics because of an ARI between November 1995 and April 1998 were used for a first clinical evaluation. Detection of one of the microorganisms included in the assay was achieved for 395 of 1,118 (35%) clinical samples, of which 37.5% were RSV, 20% were influenza A virus, 12.9% were adenovirus, 10.6% were enterovirus, 8.1% were M. pneumoniae, 4.3% were parainfluenzavirus type 3, 3.5% were parainfluenzavirus type 1, 2.8% were influenza B virus, and 0.2% were C. pneumoniae. Seasonal variations in the rates of detection of the different organisms were observed, as was expected from the literature. The levels of concordance with the data obtained by commercially available enzyme immunoassays were 95% for RSV and 98% for influenza A. The results show that the multiplex RT-PCR–enzyme-linked immunosorbent assay is a useful and rapid diagnostic tool for the management of children with ARI. Studies of the overall benefit of this method with regard to the use of antibiotics, the use of diagnostic procedures including additional microbiological tests, and hospitalization rate and duration are warranted.

Acute respiratory tract infections (ARIs) are the most common causes of childhood morbidity and mortality worldwide, accounting for about 30% of all childhood deaths in the developing world (19). While rarely causing death in industrialized countries, ARIs cause enormous direct and indirect health care costs (4, 10, 35). The causative agents of ARIs encompass a wide variety of microorganisms. Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis (2) are the most common bacteria encountered. As commensal organisms of the upper respiratory tract they usually contaminate sputum samples or nasopharyngeal aspirates or swabs, and thus, their etiological role in ARIs is difficult to prove by upper respiratory tract sampling unless invasive techniques (lung puncture) are used (24, 34).

In contrast to these bacteria, detection of Mycoplasma pneumoniae and Chlamydia pneumoniae and also the detection of viruses in a child with respiratory symptoms are usually considered evidence of acute infection. Current methods for the identification of these agents include cell culture, rapid antigen detection assays, serology (indirectly), and recently, PCR (31, 34). Cell culture techniques require specialized laboratories and are expensive, time-consuming, and labor-intensive. Rapid antigen assays are available for only a few microorganisms (influenza A virus and respiratory syncytial virus [RSV] in most countries). Serology usually requires documentation of a rise in antibody concentration from an acute-phase to a convalescent-phase blood sample, and thus, test results come in too late to be of relevance for the treatment of acute disease. While no rapid method for the microbiological diagnosis of ARI is available for routine use, the availability of such a test could result in less antibiotic therapy and more precisely tailored antibiotic therapies, resulting in reduced costs, fewer side effects, and a reduction of the emergence of resistance (38).

Currently available nucleic acid amplification techniques such as PCR (29) and reverse transcription (RT)-PCR (RT-PCR) are highly sensitive techniques for the detection of nucleic acid sequences from viruses and bacteria in clinical specimens (21, 30). These amplification techniques are particularly advantageous for the detection of fastidious or difficult-to-culture organisms such as RSV (25) or M. pneumoniae (37). Previous studies with PCR and RT-PCR for the diagnosis of ARIs have focused on the detection of a single virus or bacterium; however, the diagnostic utility of nucleic acid amplification techniques for a single infectious agent is limited by the inability to establish a specific etiology whenever the result is negative and by the inability to document simultaneous infections involving more than one infectious organism. Published multiplex PCR assays for the simultaneous detection of pathogens (15, 16, 23) and multiplex RT-PCR (m-RT-PCR) assays (6, 7, 36) have included only two or three different organisms. The application of an RT-PCR panel to respiratory specimens, as described by Gilbert et al. (13), has the disadvantage of requiring different and time-consuming assay conditions for each organism detected and the use of several tubes for one sample, thus enlarging the risk of cross contamination.

Our strategy to overcoming these limitations was to use an m-RT-PCR protocol that allows the simultaneous detection, within 1 working day, of respiratory pathogens including RNA viruses (enteroviruses, influenza A and B viruses, parainfluenzavirus type 1 [PIV-1] and PIV-3, and RSV), a DNA virus (adenovirus), and bacteria (C. pneumoniae and M. pneumoniae) that do not usually colonize the upper respiratory tracts of children.

(This study was presented in part at the 20th International Congress of Chemotherapy, Sydney, Australia, 29 June 1997, and at the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, Calif., 24 to 27 September 1998.)

MATERIALS AND METHODS

Patient samples.

Nasopharyngeal aspirates were obtained from children hospitalized with ARI at our institution between November 1995 and April 1998 with a commercially available suctioning device (UnoPlast A/S, Hundested, Denmark). Diagnoses included pneumonia, wheezing bronchitis, bronchitis, laryngotracheitis (with the latter encompassing laryngitis, laryngotracheobronchitis, and spasmodic croup), pharyngitis, tonsillitis, rhinitis, conjunctivitis, and otitis media and were obtained from the computer-based discharge-diagnosis database of the hospital. While specimen collection was not complete during the first winter season (November 1995 to April 1996), it was >95% complete for the remaining seasons (i.e., starting on 1 October 1996). Specimens were collected on the first working day following hospitalization, brought directly to the laboratory, and processed immediately (during working hours) or stored at 4°C for a maximum of 24 h (after working hours) or frozen at −70°C until they were processed. Samples were split with appropriate precautions to avoid contamination, and one portion was used directly for the detection of RSV and influenza A virus antigen by the use of enzyme immunoassays (EIAs) (Becton Dickinson, Heidelberg, Germany). A second portion was used for m-RT-PCR followed by agarose gel electrophoresis and specification in a microwell hybridization analysis (m-RT-PCR–enzyme-linked immunosorbent assay [m-RT-PCR–ELISA]).

Nucleic acid extraction.

Samples received from November 1995 through July 1997 were prepared as follows. Total nucleic acids were obtained from 100 μl of respiratory specimens diluted with 100 μl of a 0.9% NaCl solution. Sodium dodecyl sulfate was added to a final concentration of 0.1%. Nucleic acid extraction was accomplished once with 1 volume of a 1:1 phenol-chloroform mixture and once with 1 volume of chloroform, and nucleic acid was precipitated with 0.3 M ammonium acetate and ethanol. Nucleic acid pellets were dried and resuspended in 15 μl of diethylpyrocarbonate-treated, bidistilled water. Specimens received from August 1997 to April 1998 were prepared with the Boehringer High Pure Viral Nucleic Acid Kit following the instructions of the manufacturer (Boehringer Mannheim, Mannheim, Germany).

Controls for the preparation procedure were as follows. One negative sample (sputa from healthy persons) was included in each series of 5 to 10 samples to monitor for potential cross contamination. In case of a false-positive result for the negative control, the m-RT-PCR was repeated for all positive clinical samples in that series with another portion of the clinical specimen. Positive controls from culture (influenza A virus, influenza B virus, PIV-1, PIV-3, or RSV) were used in each test to document the efficiency of the preparation procedure. Prepared samples were used immediately for m-RT-PCR, and the remaining aliquots were stored at −70°C.

RT-PCR.

Target sequences were regions of the F1 subunit of the fusion glycoprotein gene for RSV (primer 1, 5′-TGT TAT AGG CAT ATC ATT GA-3′; primer 2, 5′-TTA ACC AGC AAA GTG TTA GA-3′), the hemagglutinin-neuraminidase gene for PIV-1 (primer 1, 5′-CAC ATC CTT GAG TGA TTA AGT TTG ATG A-3′; primer 2, 5′-ATT TCT GGA GAT GTC CCG TAG GAG AAC-3′), the 5′ noncoding region of the PIV-3 fusion protein gene (primer 1, 5′-TAG CAG TAT TGA AGT TGG CA-3′; primer 2, 5′-AGA GGT CAA TAC CAA CAA CTA-3′), the nucleotide sequence of the 16S rRNA for M. pneumoniae (primer 1, 5′-AAG GAC CTG CAA GGG TTC GT-3′; primer 2, 5′-CTC TAG CCA TTA CCT GCT AA-3′), the nucleotide sequence of the 16S rRNA for C. pneumoniae (primer 1, 5′-TGA CAA CTG TAG AAA TAC AGC-3′; primer 2, 5′-CGC CTC TCT CCT ATA AAT-3′), a highly conserved 5′ noncoding region for enterovirus (primer 1: 5′-ATT GTC ACC ATA AGC AGC CA-3′; primer 2, 5′-TCC TCC GGC CCC TGA ATG CG-3′), nonstructural protein genes from influenza A virus (primer 1, 5′-AAG GGC TTT CAC CGA AGA GG-3′; primer 2, 5′-CCC ATT CTC ATT ACT GCT TC-3′) and influenza B virus (primer 1, 5′-ATG GCC ATC GGA TCC TCA AC-3′; primer 2, 5′-TGT CAG CTA TTA TGG AGC TG-3′), and the sequence of the hexon gene for adenoviruses (primer 1, 5′-GCC GAG AAG GGC GTG CGC AGG TA-3′; primer 2, 5′-ATG ACT TTT GAG GTG GAT CCC ATG GA-3′). Sequences were selected from procedures described previously (3, 9, 11, 18, 20, 25, 27, 37).

A total of 5 to 6 μl of the nucleic acid preparations from clinical specimens was included in the RT reaction in a final volume of 20 μl. The RT was performed for 60 min at 37°C in a buffer with the following composition: 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1 mM (each) deoxynucleoside triphosphates (Pharmacia Biotech, Uppsala, Sweden), 0.2 μg of a hexanucleotide mixture (Boehringer Mannheim) per μl, 20 U of RNAsin (Promega, Madison, Wis.), and 10 U of Moloney murine leukemia virus reverse transcriptase (Eurogentec, Seraing, Belgium).

After heat inactivation of the reverse transcriptase at 90°C for 5 min, the entire 20-μl RT reaction mixture was used for multiplex PCR in a total volume of 80 μl. The buffer composition (without consideration of the volume of the RT buffer) was 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 0.2 mM (each) dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.01 mM digoxigenin-11-dUTP (Boehringer Mannheim), 1 μM (each) primer (Eurogentec), and 5 U of AmpliTaq-Gold polymerase (Perkin-Elmer, Branchburg, N.J.). PCR was performed on a PE 9600 Thermocycler (Perkin-Elmer) as follows: 40 cycles of denaturation at 94°C for 30 s (10 min during cycle 1), annealing at 50°C for 30 s, and extension at 72°C for 30 s (7 min during cycle 40).

As a negative control, blank reagent that contained H2O was used instead of nucleic acid. As a positive control for each m-RT-PCR run, total cellular nucleic acid extracted from virus and/or bacterial stocks was used as a template in the identical master mixture which was used for the clinical specimens.

Failure of detection of template due to specimen-specific inhibitors could not be excluded; however, this effect has been shown to occur in only 1.9 to 2.5% of specimens from the respiratory tract (33).

Prevention of carryover contamination.

To prevent carryover contamination within the laboratory, the following precautions were taken. All purchased reagents were split into small aliquots. The preparation of PCR reagents, the extraction of nucleic acids from clinical samples, and the amplification step were conducted in three different rooms. Tips equipped with sealing filters (SAFESEAL tip from BIOzym, Hessisch, Oldendorf, Germany) were used for pipetting of the reagents introduced into the PCR, and all areas and equipment were decontaminated with sodium hypochlorite and Bacillol (an alcoholic disinfectant from Bode Chemie, Hamburg, Germany) prior to and after pipetting.

Agarose gel electrophoresis.

Electrophoretic separation of PCR products (10 μl) was performed for 30 min at 130 to 160 mA on 2% agarose gels in 0.5× TBE buffer (0.045 M Tris-borate, 0.001 M EDTA) and stained with ethidium bromide, and PCR products were visualized by UV illumination as described by Sambrook et al. (32). For control of fragment lengths, 0.6 to 0.8 μg of MspI-digested pUC19 DNA (MBI Fermentas GmbH, St. Leon-Rot, Germany) was applied as a marker.

Microwell hybridization analysis.

Microwell hybridization analysis was performed with the PCR-ELISA system from Boehringer Mannheim. Nine wells of a streptavidin-coated microtiter plate were each filled with 5 μl of PCR product, and the product was denatured by adding 25 μl of 0.2 N NaOH to each well. After 5 min, 200 μl of hybridization buffer containing 2 pmol of the respective biotinylated capture probe was added. The capture probes used were specific for the amplified target sequences (see above). The sequences are 5′-GAA ACA CGG ACA CCC AAA GTA-3′ for enterovirus, 5′-GTC CTC ATC GGA GGA CTT GAA TGG AAT GAT-3′ for influenza A virus, 5′-GTC AAG AGC ACC GAT TAT CAC-3′ for influenza B virus, 5′-TAC CTT CAT TAT CAA TTG GTA AGT CAA TAT ATG-3′ for PIV-1, 5′-CTC GAT GAC GCC GCG GTG C-3′ for adenovirus, and 5′-ACT CCT ACG GGA GGC AGC AGT A-3′ for M. pneumoniae. These sequences were identical to previously reported sequences (3, 9, 18, 27, 37). The sequences of the probes used for the other microorganisms are 5′-CCT GCA TTA ACA CTA AAT TC-3′ for RSV, 5′-TCT TGC TAC CTT CTG TAC TAA-3′ for C. pneumonia, and 5′-AAA ATT CCA AAA GAG ACC GGC-3′ for PIV-3. All capture probes were 3′ biotinylated and purchased from Eurogentec. Capture was allowed to proceed for 1 h at 37°C, and afterward the wells were washed four times with 200 μl of washing solution (Boehringer Mannheim) at room temperature. To each well 200 μl of anti-digoxigenin-peroxidase (10 mU/ml; Boehringer Mannheim) diluted 1/1,000 in a buffer containing 100 mM Tris-HCl and 150 mM NaCl (pH 7.5) was added. The plates were incubated for 30 min at 37°C, and the wells were washed four times with washing solution. A total of 200 μl ABTS substrate solution (Boehringer Mannheim) was added, and the wells were incubated for 30 min at 37°C. The optical density (OD) was read on a DIAS reader (Dynatech Laboratories, Guernsey, Channel Islands) at 405 nm (reference filter, 492 nm). The run was considered valid if all negative control values were less than 0.2 OD units and the positive control value was greater than 1.0 OD unit. Samples were classified as PCR positive or negative according to a cutoff OD value of 0.5 and by comparison with the results from gel electrophoresis (positive only if at least a weak band was visible on the gel). Samples with initial readings of between 0.2 and 0.5 were considered borderline and were classified as positive or negative only after retesting with the specific single primer set. Positive hybridization controls were included in each microwell hybridization assay. They consisted of PCR products derived from the positive controls that were included in the m-RT-PCR.

Administration of data.

All data obtained were managed in a Microsoft Access database. This database included all available information about patients as well as all diagnostic data and results from the m-RT-PCR–ELISA; for influenza A virus and RSV the database also included the results of the EIA.

Bacterial and viral stocks.

The bacterial and viral stocks used as positive controls were kindly provided by the following persons: B. Schweiger and E. Schreier (Robert-Koch-Institute, Berlin, Germany) for enteroviruses, influenza A virus, and influenza B virus; K. M. Edwards (Vanderbilt University, Nashville, Tenn.) for RSV, PIV-1, and PIV-3; A. Strecker (Institute for Virology, Bochum, Germany) for RSV Long and PIV-3; and R. Krausse and P. Rautenberg (Institute for Medical Microbiology, Kiel, Germany) for M. pneumoniae, C. pneumoniae, and adenoviruses. The exact numbers of viruses or bacteria within these samples were not known. For preliminary sensitivity testing of the m-RT-PCR, consecutive dilutions (10-fold steps) of nucleic acids prepared from these cultures were used as templates for the m-RT-PCR and for amplification with single primer sets. The probable amount of nucleic acids which was sufficient to result in an amplification product was calculated on the basis of an assumed particle number (108/ml of undiluted sample) which was not confirmed by another test. To detect possible cross-reactivity among the organisms, 1 μl of undiluted nucleic acid from each organism was used in an m-RT-PCR.

RESULTS

m-RT-PCR with bacterial and viral nucleic acids.

The m-RT-PCR–ELISA procedure was tested with nucleic acids prepared from the stock solutions as described in Materials and Methods. As can be seen in Fig. Fig.1,1, only one specific amplification product could be observed in each lane. The predicted sizes of the amplification products (C. pneumoniae, 463 bp; M. pneumoniae, 277 bp; influenza B virus, 249 bp; RSV, 239 bp; PIV-3, 205 bp; influenza A virus, 190 bp; PIV-1, 179 bp; enterovirus, 154 bp; adenovirus, 134 bp) were in good agreement with the fragment sizes calculated from the agarose gel (Fig. (Fig.1).1). This suggests that the m-RT-PCR yielded specific products. However, differentiation of influenza A virus and PIV-3 as well as of influenza B virus and RSV by fragment size in gel electrophoresis alone is difficult, but the absorbance values obtained by the PCR-ELISA confirmed this specificity. Unconsumed primers are visible at the bottom of the gel.

FIG. 1
Separation of m-RT-PCR products on an agarose gel. A total of 10 μl of each of the m-RT-PCR products was separated on a 2% agarose gel. The m-RT-PCR was performed with 1 μl of viral or bacterial nucleic acid as the template, as ...

In order to estimate the sensitivity of the m-RT-PCR, nucleic acids prepared from virus stock solutions were diluted consecutively in 10-fold steps and were tested with specific primer pairs to produce the amplification products that were visible on the agarose gel and that were specified in the PCR-ELISA. Assuming that a maximum of 108 of the respective microorganisms per ml were present in the original stock solution, it was calculated that the method was able to detect 1 target sequence of M. pneumoniae and 1 target sequence of C. pneumoniae, 10 target sequences of adenovirus DNA and enterovirus RNA, and 100 target sequences of PIV-1, PIV-3, influenza A virus, influenza B virus, and RSV RNA in the analogs from the m-RT-PCR.

Comparison of EIA with m-RT-PCR–ELISA.

To receive information about the quality of the m-RT-PCR–ELISA, we compared the results obtained by m-RT-PCR–ELISA with those obtained by commercial EIAs. By this EIA 940 clinical specimens were tested for the presence of influenza A virus and 1,031 clinical specimens were tested for the presence of RSV. The results are summarized in Table Table1.1. The overall accordance of the PCR results for RSV to those obtained by EIA was 95% (with 140 positive specimens and 891 negative specimens in EIA used as a reference [100%]). Twenty-five specimens were identified as RSV positive by PCR-ELISA only, and 24 were identified as RSV positive by EIA only. In the case of influenza A virus, the overall accordance of the PCR results to those obtained by EIA was 98% (with 53 positive specimens and 887 negative specimens in the EIA used as a reference [100%]). One specimen was positive by EIA only, and 14 specimens were positive by PCR-ELISA only.

TABLE 1
Comparison of EIA and m-RT-PCR–ELISA

Results of m-RT-PCR–ELISA with clinical specimens.

A total of 1,118 samples were tested by m-RT-PCR–ELISA. The number of samples tested over time and the proportion of samples with positive PCR results can be taken from Fig. Fig.2.2. The amount of specimens increased periodically during all cold seasons (from November to April), and this correlated with an increased number of samples with positive PCR results. During the winter season of 1996–1997 the maximum number of patient samples (n = 106) was received in February, and detection of at least one microorganism by m-RT-PCR was accomplished for 48% of the samples. The lower number of specimens in the winter of 1995–1996 was due to incomplete sample collection early during our test series. Results for the different microorganisms are shown in detail in Fig. Fig.3.3. A total of 723 (65%) specimens were negative and 395 (35%) specimens were positive for at least one of the organisms included in the test. Of the isolates, 37.5% were RSV, 20.0% were influenza A virus, 12.9% were adenoviruses, 10.6% were enteroviruses, 8.1% were M. pneumoniae, 4.3% were PIV-3, 3.5% were PIV-1, 2.8% were influenza B virus, and 0.2% were C. pneumoniae (on the basis of all specimens being positive by m-RT-PCR–ELISA). RSV and influenza A were mainly detected from December to May. For influenza B virus (February to April 1997) and for PIV-1 (September to December 1997) only one main peak was observed. Infections with adenovirus, enterovirus, PIV-3, and M. pneumoniae were detected more or less constantly over the time period studied. C. pneumoniae was detected only once, in January 1997.

FIG. 2
Proportion of positive m-RT-PCR results. The number of samples with positive m-RT-PCR results and the total number of samples are given on the y axis. The time scale on the x axis is from November 1995 to April 1998.
FIG. 3
Frequency of clinical specimens with positive m-RT-PCR–ELISA results. The number of samples with positive m-RT-PCR results for each of the nine organisms is given on the y axis. The time scale on the x axis is from November 1995 to April 1998. ...

Simultaneous detection of two organisms.

The m-RT-PCR revealed evidence of simultaneous infection with two organisms in 20 specimens (1.8% of the total or 5% of the positive specimens). Coamplification of the adenovirus nucleic acid sequence occurred with C. pneumoniae (once), enterovirus (once), influenza A virus (once), and RSV (five times). Dual infections involving enteroviruses were detected with adenovirus (once), influenza A virus (three times), influenza B virus (once), PIV-3 (three times), M. pneumoniae (once), and RSV (three times). Furthermore, influenza B virus nucleic acid was coamplified with RSV and M. pneumoniae with PIV-1 from one specimen each.

Clinical data.

Clinical data were available as of February 1995 for 861 of 1,061 sample-patient pairs, with second or follow-up samples from the same hospital admission of one patient being excluded. Among these 861 patients, 550 were between 0 and 2 years of age, 153 were between 2 and 5 years of age, and 158 were older than 5 years of age. In 62% of those specimens, no bacterial or viral nucleic acids could be detected by m-RT-PCR. The most frequent diagnosis in this hospital-based study was pneumonia (309 cases [36%]). It was most commonly caused by RSV (n = 59), influenza A virus (n = 17), M. pneumoniae (n = 16), and adenovirus (n = 15). Enterovirus, PIV-3, PIV-1, and influenza B virus were associated with fewer than 10 pneumonia cases each. Among 167 patients with wheezing bronchitis (19% of 861 specimens), RSV was detected in 45 patients, adenovirus was detected in 16 patients, and enteroviruses, influenza A virus, influenza B virus, PIV-1, PIV-3, and M. pneumoniae were detected in fewer than 10 patients each. Bronchitis was observed in a total of 95 patients (11% of the 861 specimens); and the detected organisms were RSV (13 patients), enterovirus and influenza A (4 patients each), and adenovirus (3 patients). Among the 861 specimens in which a microorganism was detected by m-RT-PCR in fewer than 10 specimens, rhinitis was diagnosed in 7.1%, laryngotracheitis was diagnosed in 6.2%, and pharyngitis, otitis media, tonsillitis, and conjunctivitis were diagnosed in fewer than 5% each. Other diseases were detected in 9.1% of the patients.

The frequency of detection of one of the nine organisms in the PCR for a given respiratory disease is shown in Fig. Fig.4.4. RSV was most commonly associated with pneumonia, wheezing bronchitis, bronchitis, otitis media, or pharyngitis. Influenza A virus was associated with more than 5% of the cases of otitis media, tonsillitis, pharyngitis, laryngotracheitis, and pneumonia; enteroviruses were associated with more than 5% of cases of tonsillitis and pharyngitis; adenoviruses were associated with pharyngitis, wheezing bronchitis, conjunctivitis, and tonsillitis; M. pneumoniae was most commonly associated with pneumonia; PIV-1 was mainly associated with laryngotracheitis; and PIV-3 was associated with laryngotracheitis and conjunctivitis. C. pneumoniae was detected only once, in a patient with bronchitis.

FIG. 4
Percentage of organisms causing infections. The proportions of organisms causing a respiratory disease are given as a percentage of the total number of patients infected with organisms causing the disease. Organisms not included in the test are not indicated ...

DISCUSSION

In order to provide a technique for the rapid detection of a wide array of noncolonizing microorganisms causing ARIs, we developed and tested a single-tube m-RT-PCR–ELISA. By this technique we were able to detect nine different pathogens of the respiratory tract within 1 working day. The organisms were chosen to allow the clinician appropriate treatment choices: no antibiotic treatment for viral infections or erythromycin for mycoplasma and chlamydia infections.

Previously described primer pairs and probes from conserved regions were used in the amplification of specific products. To assess the integrity of the primer pairs and probes in this m-RT-PCR assay, RNA and DNA from viral and bacterial stocks were assayed in the presence of all nonhomologous primer pairs, and no cross-reactions were detected, documenting the high degree of specificity of this assay. When primer pairs were used in the multiplex approach, the calculated sensitivity with dilutions of prepared nucleic acids was satisfactory (detection limit, at least 1 to 100 copies).

The primer pair used for C. pneumoniae is described to react specifically with this organism, while Chlamydia psittaci and Chlamydia trachomatis DNAs do not serve as templates. The primer pair is capable of detecting 6 diverse C. pneumoniae strains but shows no cross-reaction with a set of 15 other bacterial strains tested (11). The species-specific M. pneumoniae primer pair allows detection of at least one target sequence in the m-RT-PCR approach, as has also been described for the species-specific M. pneumoniae single primer pair by van Kuppeveld et al. (37). In the case of adenovirus, the primers and probes used here are from a region coding for the hexon gene and have a high degree of homology with nucleic acids from at least 47 human adenovirus serotypes tested, while they do not detect other respiratory viruses (18). The primer pairs selected for influenza A and influenza B viruses are highly type specific for all subtypes of influenza virus (3) and show no cross-reactivity with a great variety of other respiratory microorganisms. Amplification of enterovirus nucleic acids with the enterovirus primer pair used is specific (27) and encompasses at least 27 of the isolated serotypes of this virus (28). For the primer pair used for PIV-1 a high degree of specificity was shown by Fan and Henrickson (9), with no cross-reactivity with RSV, measles virus, or an influenza virus, and (in comparison) was observed to have a good sensitivity by our m-RT-PCR assay. For RSV the complementary regions of the primer pair within the fusion glycoprotein gene are identical for both subgroups A and B of RSV, and no cross-reactivity could be detected with isolates of adenovirus, PIV-1, PIV-3, influenza A virus, or influenza B virus (25).

Thus, the data available from the literature and the results presented here indicate that the primer pairs and probes used are highly specific and sensitive, although for some organisms the sensitivity was reduced by up to a factor of 10 in the m-RT-PCR approach. This is probably due to the accumulation of by-products through unspecific annealing during PCR if the annealing temperature is suboptimal for some of the templates. The specificity in the m-RT-PCR–ELISA is supposedly the same as that with PCR amplification using the single primer pair, and therefore, m-RT-PCR is a powerful tool for the simultaneous detection of the organisms described here. In addition, compared to commercially available rapid EIAs, which are not available for most organisms, m-RT-PCR gave adequate results with an accordance of more than 95%.

In the experiments performed with clinical specimens, it was possible to test all specimens simultaneously for the presence of nucleic acids from nine microorganisms with comparatively little effort, thus making this method well-suited for use in epidemiological studies as well as for rapid microbiological studies in the clinical setting. While the samples available for testing were incomplete for the first winter season (1995–1996), more complete data from the following winter seasons gave a first insight into the epidemiology of ARI in children admitted to our hospital. RSV was the most common cause of pneumonia and bronchitis; adenoviruses and enteroviruses were observed throughout the year, with a small peak burst for adenovirus in May 1997 and also in March 1998. Influenza A virus peaks were observed mainly in the winter months. Interestingly, we observed only one peak for influenza B virus (in 1997), which occurred about 4 weeks later than the influenza A virus peak. The incidence of PIV-1 was very low from the beginning of this study in November 1995 until August 1997, whereas from September to December 1997 a total of 12 cases were detected, possibly reflecting a community outbreak, as reported by others to occur every 2 to 3 years (22).

The low detection rate for C. pneumoniae was surprising. Using the PCR technique, Falck et al. (8) had suggested that C. pneumoniae is present in up to 5.7% of healthy children and in up to 45% of children with upper respiratory tract infection, whereas Goo et al. (14) showed that C. pneumoniae was rare in young children with otitis media. In the experiments of Falck et al. (8), swabs instead of nasopharyngeal aspirates were used, and a more vigorous swabbing might have resulted in a better recovery rate of cells which are the habitats for C. pneumoniae. Such effects were described in a study in which the efficiency of detection of M. pneumoniae was tested with regard to different methods of specimen collection (26). Also, contaminating ingredients (ions, inhibitors) may have a negative influence on the specific amplification of C. pneumoniae target sequences. However, as described by Stauffer et al. (33), such inhibition of PCR occurs only with 1.9 to 2.5% of specimens from the respiratory tract. Additional studies are needed to clarify this observation.

Evidence of simultaneous infection in children with ARI was observed by m-RT-PCR, and the simultaneous infections were most frequently with adenoviruses and enteroviruses, as expected. Simultaneous infections have also been observed by others who used a panel of PCR techniques (13) or bacterial and viral serology (12). In the latter study, multiple infections were seen in 8% of the patients studied, whereas 1.8% of all samples (5.0% of positive samples) observed here had multiple infections (tests for the encapsulated colonizing bacteria were excluded from our study). In a retrospective review, the percentage of dual respiratory viral infections increased proportionally with the number of diagnostic methods used (5), and it was up to 11.6% when PCR, cell culture, and serology were used together.

Overall, the results of our 3-year evaluation of m-RT-PCR correspond very well with published data on the epidemiology (17) and spectrum of disease of those organisms detected by the method. In addition, as clinically observed by one of us, the usefulness of the method was tremendous, resulting in fewer prescriptions of antibiotics, shortened hospitalization periods, and lower costs for other diagnostic procedures including microbiological tests. An overall, prospective evaluation of the clinical and cost benefits of m-RT-PCR is warranted (1).

ACKNOWLEDGMENTS

We are indebted to our colleagues who kindly provided us with microorganisms which served as positive controls for our test: B. Schweiger, E. Schreier, K. M. Edwards, A. Strecker, R. Krausse, and P. Rautenberg. The excellent technical assistance of B. Thomsen, S. Rockahr, and T. Rücker is greatly appreciated.

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