Multiplex PCR for rapid and differential diagnosis of Mycoplasma pneumoniae and Chlamydia pneumoniae in respiratory infections

doi:10.1016/S0732-8893(99)00070-X

Diagnostic Microbiology and Infectious Disease

Volume 35, Issue 2, October 1999, Pages 105-108

Presented in part at the Meeting of the Italian Society for the Study of Infectious and Parasitic Diseases, Tuema Section, Certosa di Pontignano (Siena), Italy, June, 13, 1998.

https://doi.org/10.1016/S0732-8893(99)00070-X Get rights and content

Abstract

A duplex polymerase chain reaction (PCR) was developed for the simultaneous detection of Chlamydia pneumoniae and Mycoplasma pneumoniae. A study of 163 respiratory specimens from in-patients of the “Centre Hospitalier et Universitaire de Nancy” showed the good sensitivity of this duplex PCR allowing the detection of C. pneumoniae and M. pneumoniae from 8 and 13 patients, respectively, whereas the culture was negative for C. pneumoniae for all the samples and positive for M. pneumoniae only in 9 cases. The value of these results has been confirmed by running on the same samples specific nested PCRs for these two microorganisms that gave the same results. Thus, the proposed duplex amplification technique may facilitate the diagnosis of infection by these two agents that are difficult to isolate.

Introduction

Chlamydia pneumoniae and Mycoplasma pneumoniae are responsible for community-acquired respiratory tract infections, in particular atypical pneumonia Clyde 1993, Kuo et al 1995. M. pneumoniae infections are acute and may require hospitalization, whereas C. pneumoniae infection, frequently asymptomatic, can be responsible for long lasting diseases, more severe in elderly patients (Troy et al. 1997). For a specific treatment to be administered a rapid laboratory diagnosis must be obtained. Isolation of M. pneumoniae and C. pneumoniae requires 6 to 21 days and is often negative. Rapid laboratory tests such as antigen detection or hybridization are of limited sensitivity and serology is often unconclusive. Thus, gene amplification that permits a rapid and sensitive diagnosis is the method of choice Ieven and Goossens 1997, Whelen and Persing 1996. When different agents are searched for, a multiplex polymerase chain reaction (PCR) is of interest Cadieux et al 1993, Valassina et al 1997. Thus we have devised such a method for detection of C. pneumoniae and M. pneumoniae and applied it to 163 clinical samples from in-patients with respiratory symptoms of Nancy University Hospital.

Section snippets

Clinical samples and cultures

During the winter-spring 1998 season, 129 broncho-alveolar lavages (BALs), 20 bronchial aspirates, 12 pharyngeal swabs, and 2 sputa were collected from 163 in-patients of the Centre Hospitalier Universitaire of Nancy, France. All patients presented with respiratory symptoms and were aged from 1 to 81. An aliquot (200 μL) of the clinical sample was used for isolation of C. pneumoniae onto HL cells infected in triplicate (Kuo and Grayston 1990). A blind passage was performed in duplicate five

Results

Multiplex PCR, as well as single PCR with the same primers, detected as little as 2 CFU of M. pneumoniae and 2 IFU of C. pneumoniae whatever the sample tested: extracted DNA from buffer dilution or from simulated clinical samples (data not shown). The specificity of the PCR reaction was satisfactory as no aspecific amplification was noted with any of the tested pathogens (Fig. 1). All 163 DNA extracts proved amplifiable as primers for γ-interferon gave the expected amplicon with all extracts

Discussion

As expected, PCR proved more sensitive than culture for the diagnosis of M. pneumoniae and C. pneumoniae infections. For M. pneumoniae, antibiotic therapy or inadequate management of samples may explain the low isolation rate. Isolation of C. pneumoniae is seldom obtained and its isolation from elderly patients is almost never achieved (Kuo et al. 1995). Thus, a negative result of culture is not surprizing. Recently, Gröndahl et al. (1999) presented a multiplex RT-PCR protocol detecting up to

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The chosen primers of M pneumoniae enclosed a specific 144 bp fragment: MP5-1 (5′-GAA GCT TAT GGT ACA GGT TGG-3′) and MP5-2 (5′-ATT ACC ATC CTT GTT GTA AGG-3′).11 The primers for C pneumonia—specific for the Omp1 gene—were: Cpn1 (5′-GTT CAA TCT CGT TGG TTT ATT-3′, nt 453–473) and Cpn3 (5′-TCC AAT GTA TGG CAC TAA AGA-3′, nt 858–838, 405 bp).12 Every sample with a negative result using multiplex PCR was reanalyzed using specific nested PCRs under the same conditions to differentiate between the two pathogens.
Atypical bacterial pathogens—including Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Chlamydia trachomatis—are important infectious agents of the respiratory system. Most current information pertains to adults and little is known about the role of these organisms in lower respiratory tract infections among young children with acute bronchiolitis.
This study detected these pathogens in the nasopharyngeal secretions of children between 1 month and 2 years of age admitted with acute bronchiolitis to hospitals in Khon Kaen, northeast Thailand. The M pneumoniae and C pneumoniae in the nasopharyngeal secretions were detected using multiplex and nested-polymerase chain reaction (PCR), whereas PCR and restriction fragment length polymorphism were used to investigate C trachomatis. These samples were also tested by multiplex reverse transcriptase PCR for respiratory viruses, including respiratory syncytial virus (RSV), influenza A, influenza B, and human metapneumovirus.
Of the 170 samples taken from hospitalized children with acute bronchiolitis, 12.9% were infected with atypical bacteria and 85.3% with respiratory viruses. RSV was the most common causative viral agents found in 64.7% of the samples. M pneumoniae was the most common atypical bacterial pathogen (14/170, 8.2%) and most of the patients infected with it were between 6 and less than 12 months of age (71 cases). Of the infected cases in this age group, 7 of 14 were infected with M pneumoniae and 4 of 4 with C pneumoniae. Both M pneumoniae (13/14) and C pneumoniae (4/4) had etiologies indicating viral coinfections. Four (2.4%) of all of the cases had C trachomatis infections and all of these were infected with RSV, including three patients less than 6 months of age.
These results suggest that in children with virus-induced acute bronchiolitis coinfection with M pneumoniae, C pneumoniae, or C trachomatis can be expressed differently in each age group. These atypical bacteria may be the important infectious agents that induce severe illness of acute bronchiolitis.
Specific Diagnostic Tests for Atypical Respiratory Tract Pathogens
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Because M pneumoniae is only one of a variety of fastidious and/or slow-growing pathogenic microorganisms responsible for RTI with clinically similar manifestations, there has been considerable interest and effort to develop multiplex PCR assays for single-step detection of multiple causative agents. Most assays now include gene targets for M pneumoniae, C pneumoniae, L pneumophila, and, occasionally, other organisms.26,30–40 Still, monoplex assays seem to have higher sensitivity and specificity than multiplex assays.41
Development and evaluation of a multiplex test for the detection of atypical bacterial DNA in community-acquired pneumonia during childhood
2009, Clinical Microbiology and Infection
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Although there are numerous single PCR systems for microorganism detection, multiplex PCR protocols are less common, despite the savings that they offer in time and cost. Multiplex PCR systems are available that include some of the microorganisms investigated in the present study, e.g. M. pneumoniae and C. pneumoniae [28], C. pneumoniae, C. psittaci and M. pneumoniae [29], C. pneumoniae, M. pneumoniae and L. pneumophila [30], and C. pneumoniae, M. pneumoniae and genus Legionella [24]. Others include M. pneumoniae and C. pneumoniae with Streptococcus pneumoniae and H. influenzae [23].
An incorrect or late diagnosis can lead to an increase in the morbidity and mortality caused by pneumonia, and the availability of a rapid and accurate microbiological test to verify the aetiology is imperative. This study evaluated a molecular test for the identification of the bacterial cause of atypical community-acquired pneumonia (ACAP). Fifty-four children with pneumonia were studied using bacteriological cultures, Mycoplasma pneumoniae, Coxiella burnetii, Chlamydophila pneumoniae and Legionella spp. serology, and Streptococcus pneumoniae and Legionella antigens. Simultaneously, the presence of bacterial and fungal DNA was tested for in respiratory secretion samples using the Vircell SL kit, including multiplex PCR and amplicon detection by means of line blots. There were 14 cases of ACAP caused by M. pneumoniae, with positive kit results for 13 of them, and two cases of Q-fever, with negative kit results for Coxiella burnetii. The test was negative in the remaining 38 cases (one staphylococcal pneumonia, 20 Streptococcus pneumoniae pneumonias, and 17 probable viral pneumonias). The sensitivity of the test for the detection of M. pneumoniae was 92.8% and the specificity was 100%. The Vircell SL kit allows detection of M. pneumoniae DNA in respiratory secretion samples from children with ACAP.
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Classic findings on chest radiographs include subsegmental atelectasis and interstitial infiltrates. A diagnosis may be made on clinical grounds, whereas polymerase chain reaction (PCR), culture, and serology can be used to confirm the diagnosis.13,14 Oral treatment with macrolides, such as erythromycin and clarithromycin, is usually sufficient.
Pneumonia is an important clinical problem that affects children of all ages. Although effectively treated on an outpatient basis in the majority of cases, some children with respiratory infections still require hospitalization. This may be particularly true for patients with immunocompromise, for whom the lung represents the most common site of infection. Furthermore, respiratory infections represent a significant source of morbidity and mortality in this patient population. This article focuses on the clinical presentation, etiology, and treatment of childhood pneumonia, with special consideration given to the immunocompromised child. Two specific complications of pneumonia, lung abscess and empyema, are discussed.
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Chlamydia pneumoniae is established as a common agent of acute respiratory tract infection and has been implicated in the pathogenesis of asthma and chronic obstructive pulmonary disease. Airway disease is a prominent cause of morbidity and mortality after lung transplantation. We investigated the role of C pneumoniae as a pulmonary pathogen after lung transplantation.
Eighty lung transplant recipients underwent 232 bronchoscopies with bronchoalveolar lavage with or without transbronchial lung biopsy during 1 year for surveillance of rejection and infection, or where clinically indicated.
C pneumoniae was detected using nested polymerase chain reaction in 9 of 36 (25%) recipients studied within 30 days of lung transplantation, 3 of whom remained positive on repeat lavage and died from airway disease in the first year post-operatively. By comparison, all 27 recipients with negative lavage survived >1 year. Lavage was positive for C pneumoniae in 18 of 71 (25%) recipients studied >30 days after lung transplantation, 5 of whom had pneumonia and 8 of whom had bronchiolitis obliterans syndrome. Eleven also had acute pulmonary allograft rejection.
Persistent infection with C pneumoniae (whether donor-derived, de novo or re-activated) appears deleterious to pulmonary allograft function and is associated with early mortality, rejection and bronchiolitis obliterans syndrome after lung transplantation. A trial of empiric antibiotic therapy for C pneumoniae may therefore be warranted in the attempt to prevent progressive inflammatory airway disease.

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Diagnostic Microbiology and Infectious Disease

Abstract

Introduction

Section snippets

Clinical samples and cultures

Results

Discussion

References (15)

Rapid detection of different RNA respiratory virus species by multiplex RT-PCRapplication to clinical specimens

Clin Diagn Virol

Detection of Mycoplasma pneumoniae by using the polymerase chain reaction

J Clin Microbiol

Use of a triplex polymerase chain reaction for the detection and differentiation of Mycoplasma pneumoniae and Mycoplasma genitalium in the presence of human DNA

J Gen Microbiol

Clinical overview of typical Mycoplasma pneumoniae infections

Clin Infect Dis

Diagnostic utility of PCR-enzyme immunoassay, culture, and serology for detection of Chlamydia pneumoniae in symptomatic and asymptomatic patients

J Clin Microbiol

Rapid identification of nine microorganisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-PCRfeasibility study

J Clin Microbiol

Relevance of nucleic acid amplification techniques for diagnosis of respiratory tract infections in the clinical laboratory

Clin Microbiol Rev

Cited by (30)

Atypical bacterial pathogen infection in children with acute bronchiolitis in northeast Thailand

Specific Diagnostic Tests for Atypical Respiratory Tract Pathogens

Development and evaluation of a multiplex test for the detection of atypical bacterial DNA in community-acquired pneumonia during childhood

Respiratory infections: Pneumonia, lung abscess, and empyema

Infections and Diseases of the Lungs, Pleura, and Mediastinum

Chlamydia pneumoniae infection after lung transplantation

BacteriologyMultiplex PCR for rapid and differential diagnosis of Mycoplasma pneumoniae and Chlamydia pneumoniae in respiratory infections

Abstract

Introduction

Section snippets

Clinical samples and cultures

Results

Discussion

Clin Diagn Virol

Detection of Mycoplasma pneumoniae by using the polymerase chain reaction

J Clin Microbiol

Use of a triplex polymerase chain reaction for the detection and differentiation of Mycoplasma pneumoniae and Mycoplasma genitalium in the presence of human DNA

J Gen Microbiol

Clinical overview of typical Mycoplasma pneumoniae infections

Clin Infect Dis

Diagnostic utility of PCR-enzyme immunoassay, culture, and serology for detection of Chlamydia pneumoniae in symptomatic and asymptomatic patients

J Clin Microbiol

Rapid identification of nine microorganisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-PCRfeasibility study

J Clin Microbiol

Relevance of nucleic acid amplification techniques for diagnosis of respiratory tract infections in the clinical laboratory

Clin Microbiol Rev

Bacteriology
Multiplex PCR for rapid and differential diagnosis of Mycoplasma pneumoniae and Chlamydia pneumoniae in respiratory infections