Comparison and evaluation of real-time PCR,real-time nucleic acid sequence-based amplification,conventional PCR,and serology for diagnosis of Mycoplasma pneumoniae

Mycoplasma pneumoniae is a common cause of community-acquired pneumonia and lower-respiratory-tract infections. Diagnosis has traditionally been obtained by serological diagnosis, but increasingly, molecular techniques have been applied. However, the number of studies actually comparing these assays is limited. The development of a novel duplex real-time PCR assay for detection of M. pneumoniae in the presence of an internal control real-time PCR is described. In addition, real-time nucleic acid sequence-based amplification (NASBA) on an iCycler apparatus is evaluated. These assays were compared to serology and a conventional PCR assay for 106 clinical samples from patients with lower-respiratory-tract infection. Of the 106 samples, 12 (11.3%) were positive by all the molecular methods whereas serology with acute sample and convalescent samples detected 6 (5.6%) and 9 (8.5%), respectively. Clinical symptoms of the patients with Mycoplasma-positive results were compared to those of the other patients with lower-respiratory-tract infections, and it was found that the results for mean lower age numbers as well as the presence of chills, increased erythrocyte sedimentation rate, and raised C-reactive protein levels showed significant differences. Molecular methods are superior for diagnosis of M. pneumoniae, providing more timely diagnosis. In addition, using real-time methods involves less hands-on time and affords the ability to monitor the reaction in the same tube.     

Evaluation of an indirect haemagglutination kit for the rapid serological diagnosis of Mycoplasma pneumoniae infections.

A recently introduced indirect haemagglutination kit for the detection of Mycoplasma pneumoniae antibodies is compared with the standard method complement fixation. It is concluded that the kit provides a rapid, simple, and moderately sensitive means of detecting antibodies and, in some cases, enables these to be detected in the early, acute stage of infection.     

Comparison of two rapid commercial tests with complement fixation for serologic diagnosis of Mycoplasma pneumoniae infections.

The complement fixation (CF) test is the current reference serologic test for the diagnosis of Mycoplasma pneumoniae infection. However, it is reported to be insensitive and nonspecific, and it is labor intensive. To determine if a faster and more sensitive diagnosis of M. pneumoniae could be obtained, we examined 50 paired serum samples from patients with suspected M. pneumoniae infection by the CF test and two commercial rapid antibody detection kits, the Remel M. pneumoniae immunoglobulin G (IgG)-IgM antibody test system (Remel, Lenexa, Kans.) and the Seradyn Color Vue M. pneumoniae IgG-IgM kit (Seradyn, Indianapolis, Ind.). The Remel test, a 5-min qualitative immunobinding assay, detected antibodies in three patient serum samples with CF titers of 32 and in all but one sample with titers of > or = 64. The Seradyn test, a 40-min qualitative agglutination test, was less sensitive than CF or Remel. The Seradyn test was positive in 68% of cases, compared with 94 and 96% of cases tested by CF or Remel, respectively. Both commercial tests are faster and less technically demanding to perform than is the CF test.     

Evaluation of a microscopy method for rapid detection and identification of Mycoplasma pneumoniae.

A microscopy test that used the typical shape of Mycoplasma pneumoniae cells growing on glass was investigated for its value for diagnostic purposes. Suspensions from 108 throat swabs were infected artificially with 102, 103, and 104 colony-forming units of three M. pneumoniae strains per ml. Agar medium, a diphasic medium, and the microscopy method with liquid medium in cover slip chambers were compared for isolation of the mycoplasmas. The mycoplasms were detected first by the microscopy method in nearly all concentrations tested. Typical M. pneumoniae cells could often be detected after 48 h. No differences were found between a laboratory strain and two low-passage strains. The experimental results suggest that under special circumstances the microscopy method could be a useful tool for isolation and identification of M. pneumoniae.     

Detection of viral, Chlamydia pneumoniae and Mycoplasma pneumoniae infections in exacerbations of asthma in children.

BACKGROUND: A high frequency of virus infections has been recently pointed out in the exacerbations of asthma in children. OBJECTIVES: To confirm this, using conventional and molecular detection methods, and expanding the study to younger children. STUDY DESIGN: One hundred and thirty-two nasal aspirates from 75 children hospitalized for a severe attack of asthma were studied (32 infants, mean age 9.1 months; and 43 children, mean age 5.6 years). According to the virus, a viral isolation technique, immunofluorescence assays (IFA) or both were used for the detection of rhinovirus, enterovirus, respiratory syncytial (RS) virus, adenovirus, coronavirus 229E, influenza and parainfluenza virus. Polymerase chain reaction (PCR) assays were used for the detection of rhinovirus, enterovirus, RS virus, adenovirus, coronavirus 229E and OC43, Chlamydia pneumoniae and Mycoplasma pneumoniae. RESULTS: Using IFA and viral isolation techniques, viruses were detected in 33.3% of cases, and by PCR techniques, nucleic acid sequences of virus, Chlamydia pneumoniae and Mycoplasma pneumoniae were obtained in 71.9% of cases. The combination of conventional and molecular techniques detects 81.8% of positive samples. Two organisms were identified in the same nasal sample in 20.4% of the cases. The percentage of detections was higher (85.9%) in the younger group than in the other (77%). The most frequently detected agents were rhinovirus (46.9%) and RS virus (21.2%). Using PCR rather than conventional techniques, the detection rates were increased 5.8- and 1.6-fold in rhinovirus and RS virus infections, respectively. The detection levels of the other organisms are as follows: 9.8, 5.1, 4.5, 4.5, 4.5, 3.7, and 2.2% for enterovirus, influenza virus, Chlamydia pneumoniae, adenovirus, coronavirus, parainfluenza virus, and Mycoplasma pneumoniae, respectively. CONCLUSION: These results confirm the previously reported high frequency of rhinovirus detection in asthmatic exacerbations in children. They also point out the frequency of RS virus detection, and emphasize the fact that PCR assays may be necessary to diagnose respiratory infections in asthma.     

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

To assess the utility of PCR-enzyme immunoassay (EIA) for diagnosis of acute infection with Chlamydia pneumoniae, we compared tissue culture, PCR-EIA, direct fluorescent-antibody (DFA) stain, and serology in studies with 56 patients with respiratory symptoms and 80 asymptomatic persons. Thirty-five patients were positive by either culture or PCR-EIA, and 101 were negative by both assays. Thirty specimens from symptomatic patients and one from an asymptomatic patient were culture positive; 23 of these were also PCR-EIA positive. Of the eight culture-positive, PCR-EIA-negative specimens, five were DFA negative and three were DFA positive. Four additional specimens were culture negative and PCR-EIA positive; of these, three were DFA positive and one was DFA negative. When we used culture- and/or DFA-positive results as a reference or "gold standard," the sensitivity and specificity of PCR were 76.5 and 99.0%, respectively. When we used PCR- and/or DFA-positive results as the reference, the sensitivity of culture was 87.5%. On the basis of single acute serum specimens, only 8 of these 35 patients had diagnostic antibody titers. Of the asymptomatic patients, 75% had immunoglobulin G or immunoglobulin M antibody to C. pneumoniae; 15 (18.8%) of these had antibody levels considered to be diagnostic of acute infection. This multicenter study indicates that culture and/or PCR-EIA is more reliable for prompt diagnosis of C. pneumoniae infection than single-point serology alone.     

Comparison of PCR and ELISA techniques in the diagnosis of Mycoplasma pneumoniae airway infections in children

Mycoplasma pneumoniae (Mycoplasma pn.) is a common airway pathogen in childhood. Mycoplasma pn. infections appear as epidemics (every 3-7 years) and are very often the cause of community--acquired pneumonia among people being in close contact for longer period of time and specially in children, young adults and elderly. For many years, routine diagnosis of Mycoplasma infections based upon ELISA methods, which determine a level of specific Mycoplasma antibodies in class G, M, A in serum. One of the most reliable new diagnostic technique is PCR, which determines Mycoplasma pn. DNA in clinical samples. The aim of the present study was to compare PCR and ELISA techniques in the diagnosis of Mycoplasma pn. airway infections in children. Sixty two children aged 2-17 years, with symptoms of Mycoplasma pn., infection, with presence of IgM, or/and IgG against Mycoplasma pn. in serum took part in the study. All children had antibodies against Mycoplasma pn. measured by ELISA method and Mycoplasma pn. DNA in PCR. The results showed high similarity between both methods (ELISA and PCR) in samples from swab throat.     

Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples

A multiplex PCR (mPCR) was developed for simultaneous detection of specific genes for Streptococcus pneumoniae (lytA), Mycoplasma pneumoniae (P1), Chlamydophila pneumoniae (ompA), and Haemophilus influenzae (16S rRNA, with verification PCR for P6). When the protocol was tested on 257 bacterial strains belonging to 37 different species, no false negatives and only one false positive were noted. One Streptococcus mitis out of thirty was positive for lytA. In a pilot application study of 81 sputum samples from different patients with suspected lower respiratory tract infection (LRTI), mPCR identified S. pneumoniae in 25 samples, H. influenzae in 29, M. pneumoniae in 3, and C. pneumoniae in 1. All samples culture positive for S. pneumoniae (n=15) and H. influenzae (n=15) were mPCR positive for the same bacteria. In a pilot control study with nasopharyngeal swabs and aspirates from 10 healthy adults, both culture and mPCR were negative. No PCR inhibition was found in any of the mPCR-negative sputum or nasopharyngeal samples. Whether all samples identified as positive by mPCR are truly positive in an aetiological perspective regarding LRTI remains to be evaluated in a well-defined patient material. In conclusion, the mPCR appears to be a promising tool in the aetiological diagnostics of LRTI.     

Etiology of invasive bacterial infections in immunocompetent children in Korea (1996-2005): a retrospective multicenter study

The purpose of this study was to identify the major etiological agents responsible for invasive bacterial infections in immunocompetent Korean children. We retrospectively surveyed invasive bacterial infections in immunocompetent children caused by eight major pediatric bacteria, namely Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Listeria monocytogenes, and Salmonella species that were diagnosed at 18 university hospitals from 1996 to 2005. A total of 768 cases were identified. S. agalactiae (48.1%) and S. aureus (37.2%) were the most common pathogens in infants younger than 3 months. S. agalactiae was a common cause of meningitis (73.0%), bacteremia without localization (34.0%), and arthritis (50%) in this age group. S. pneumoniae (45.3%) and H. influenzae (20.4%) were common in children aged 3 months to 5 yr. S. pneumoniae was a common cause of meningitis (41.6%), bacteremia without localization (40.0%), and bacteremic pneumonia (74.1%) in this age group. S. aureus (50.6%), Salmonella species (16.9%), and S. pneumoniae (16.3%) were common in older children. A significant decline in H. influenzae infections over the last 10 yr was noted. S. agalactiae, S. pneumoniae, and S. aureus are important pathogens responsible for invasive bacterial infections in Korean children.     

Rapid-cycle PCR for detection and typing of Mycoplasma pneumoniae in clinical specimens

We designed several new primers and modified previously described species- and type-specific primers targeting the Mycoplasma pneumoniae P1 adhesin gene. Optimized thermal profiles allowed one-step or nested PCR to be completed in less than 1 h. In 10 patients with pneumonia, M. pneumoniae type 1 was identified in 3 and type 2 in 7.     

Diagnosis of Mycoplasma pneumoniae pneumonia: sensitivities and specificities of serology with lipid antigen and isolation of the organism on soy peptone medium for identification of infections.

The sensitivities and specificities of isolation and serology for detection of Mycoplasma pneumoniae infections were determined for 3,546 pneumonia patients for whom both isolation and serological data were available. Soy peptone, fresh yeast extract, horse serum-supplemented agar, and diphasic medium were employed for isolation, and the lipid antigen of M. pneumoniae was used for serodiagnosis by complement fixation. The number of M. pneumoniae colonies most frequently detected was 200 to 600 per throat swab, with a range of less than or equal to 60 to greater than or equal to 2,000. The use of diphasic medium increased the number of isolates by 26% compared with direct isolation on agar plates. The organism was isolated from 360 of 525 patients who showed fourfold or greater antibody increases in their paired sera, resulting in a sensitivity of culture of 68%. When persons with titers of greater than or equal to 32 but no fourfold increase were used as the reference, the sensitivity of culture was 58%. The combined sensitivity of the culture method for persons with serological evidence of infection (fourfold increase and titer of greater than or equal to 32) was 64%. The specificity of the culture method was 97% for the 2,527 serologically negative persons. Fourfold antibody increases were found in 360 of 674 persons with isolates of the organism, resulting in a sensitivity of 53%. An additional 247 persons showed titers of greater than or equal to 32 (without a fourfold increase), resulting in a combined sensitivity of 90% for serology with the lipid antigen for the detection of antibodies in culture-positive persons. Fourfold antibody increases were found in 6% of culture-negative persons, resulting in a specificity of 94%. The quantitative culture results provide important base-line data for the development of rapid diagnostic tests for M. pneumoniae infection.     

Detection and Confirmation of Mycoplasma pneumoniae in Urogenital Specimens by PCR

Following the isolation of Mycoplasma pneumoniae from urogenital specimens (M. Goulet, R. Dular, J. G. Tully, G. Billows, and S. Kasatiya, J. Clin. Microbiol. 33:2823–2825, 1995), a study was undertaken to confirm the observations by PCR. Specific primers directed to the P1 adhesin gene of M. pneumoniae were used. A total of 300 genital specimens were tested for M. pneumoniae and Mycoplasma genitalium by culture and PCR. Of these, 15 were positive by culture and 17 were positive by PCR for M. pneumoniae. No M. genitalium was detected in any of the specimens by either method. The present study demonstrates that PCR is sensitive and rapid compared to cumbersome culture methods and can be used to detect M. pneumoniae in urogenital specimens in a routine diagnostic laboratory.     

Simultaneous detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by use of molecular beacons in a duplex real-time PCR

A real-time PCR was designed for detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae such that each pathogen could be detected in a single tube and differentiated using molecular beacons marked with different fluorochromes. This duplex PCR, targeting the P1 adhesion gene for M. pneumoniae and the ompA gene for C. pneumoniae, was compared with two conventional PCR assays targeting the 16S rRNA gene and the ompA gene. A total of 120 clinical throat and nasopharyngeal swab samples were tested. DNA extraction was performed using an alkali denaturation/neutralization method, and real-time amplification, detection, and data analysis were performed using a Rotor-Gene 2000 real-time rotary analyzer (Corbett Life Science, Sydney, Australia). Using conventional PCR as a reference in an analysis of 120 samples, 13 of 14 samples positive for C. pneumoniae were detected by the novel real-time PCR. In an analysis of M. pneumoniae, 22 samples were positive in the conventional PCR and the novel assay detected 24 positive samples. When using the conventional PCR as a reference, sensitivity and specificity were 93% and 100%, respectively, for C. pneumoniae and 100% and 98%, respectively, for M. pneumoniae. With an overall agreement of 98.8%, this suggests that performance of the new duplex real-time PCR is comparable to that of conventional PCR.     

Demonstration by a nested PCR for Mycoplasma pneumoniae that M. pneumoniae load in the throat is higher in patients hospitalised for M. pneumoniae infection than in non-hospitalised subjects

A nested PCR protocol to detect Mycoplasma pneumoniae DNA in throat specimens was developed. An amplification control (AC) template, which is amplified by the same primers as the M. pneumoniae target sequence, was constructed. The assay allowed highly specific and sensitive detection of M. pneumoniae DNA. In all, 305 throat samples, 62 from hospitalised patients and 243 from non-hospitalised subjects, were analysed by the nested PCR. Inhibition of the PCR was observed in 20% of the samples, but was abolished after a 1 in 10 dilution. Throat samples from 5 (8%) of the hospitalised patients and from 7 (3%) of the non-hospitalised subjects were positive for M. pneumoniae DNA. To investigate the relationship between M. pneumoniae load and the severity of disease, the M. pneumoniae load in 10 throat samples from M. pneumoniae-positive subjects was assessed semi-quantitatively by application of the nested PCR to a series of limiting dilutions of nucleic acid extracted from these throat samples. The calculated M. pneumoniae load varied from 20 to 3830 cfu/ml of throat sample. The mean M. pneumoniae load in samples from the hospitalised patients was significantly higher than that in samples from the non-hospitalised subjects. The nested PCR is a useful tool to detect M. pneumoniae DNA in the throat and to study the relationship between the load of M. pneumoniae in throat samples and severity of disease due to M. pneumoniae infection.     

Diagnostic utility and clinical significance of naso- and oropharyngeal samples used in a PCR assay to diagnose Mycoplasma pneumoniae infection in children with community-acquired pneumonia

PCR assays of naso- and oropharyngeal samples among hospitalized children appear equally effective for the diagnosis of serologically confirmed community-acquired mycoplasmal pneumonia. However, the combination of results from both sites yields optimal sensitivity (57%), specificity (98%), and positive (92%) and negative (82%) predictive values when compared with Mycoplasma pneumoniae enzyme-linked immunosorbent assay.     

Real-time PCR for rapid diagnosis of entero- and rhinovirus infections using LightCycler.

BACKGROUND: PCR techniques have proved to be more sensitive than traditional cell culture in the diagnosis of enterovirus and rhinovirus infections and are widely used in clinical virus laboratories. However, PCR assays are relatively time-consuming and labor intensive, particularly if separate hybridization steps are used to confirm the specificity of positive findings. OBJECTIVES: The aim of the present study was to develop fast and sensitive real-time PCR assay, which would allow simultaneous detection of entero- and rhinoviruses and their quantification in clinical and experimental samples. STUDY DESIGN: Two real-time RT-PCR protocols were developed using LightCycler (LC) technology; SYBRGreen and hybridization probe assays. The sensitivity of these assays to detect entero- and rhinoviruses was compared with that of a traditional reference RT-PCR-hybridization assay and cell culture. All PCR protocols used the same primers amplifying the 5'-non coding region (NCR) of entero- and rhinoviruses. The LC probe assay and the reference RT-PCR used almost identical detection probes, which bind to enterovirus specific amplicons. RESULTS AND CONCLUSIONS: Both real-time PCR assays were equally sensitive as the reference RT-PCR-assay and all were more sensitive than cell culture. Both real-time assays quantified reliably the amount of the virus and took much shorter time than the reference RT-PCR. As the real-time SYBRGreen assay detects both entero- and rhinoviruses it can be used for primary screening of samples, which can be positive for either of these viruses. The real-time probe-assay can confirm the presence of enterovirus in SYBRGreen positive samples or it can be used for selective screening of enteroviruses e.g. from CSF samples.     

Comparison of PCR, Culture, and Serological Tests for Diagnosis of Mycoplasma pneumoniae Respiratory Tract Infection in Children

For diagnosis of Mycoplasma pneumoniae infection we compared two rapid tests, PCR and the immunoglobulin M immunofluorescence assay (IgM IFA), with culture and the complement fixation test (CFT), in a prospective study among 92 children with respiratory tract infection and 74 controls. Based on positivity of culture and/or CFT as the diagnostic criterion, nine patients (10%) were diagnosed with M. pneumoniae infection. All patients positive by culture were also positive by PCR. In all controls cultures, PCRs, and serological assays were negative, except in one with a positive IgM IFA. The IgM IFA had a low positive predictive value of 50%. Only a combination of PCR (seven patients) and CFT (seven patients) allowed diagnosis of all cases.     

Clinical impact of a PCR assay for rapid identification of Klebsiella pneumoniae in blood cultures

The clinical impact of a rapid PCR identification assay for Klebsiella pneumoniae in positive blood cultures was prospectively evaluated. Multivariate analysis identified the rapid PCR assay as the only significant factor in decreasing the time lapse preceding the initiation of appropriate antimicrobial therapy (hazards ratio, 3.03; confidence interval, 1.62 to 5.68; P, 0.001).