16S rRNA based polymerase chain reaction compared with culture and serological methods for diagnosis of Mycoplasma pneumoniae infection

The use of a 16S rRNA based polymerase chain reaction (PCR) for the detection of Mycoplasma pneumoniae infection was investigated. Sputum samples from 34 patients with respiratory illness and evidence of pneumonia as judged by chest X-ray were analyzed by PCR and microbiological culture. Throat swabs from 14 healthy individuals were used as controls. For serology, an enzyme immunoassay for the detection of immunoglobulin M antibodies and a complement fixation assay were performed. Evidence of Mycoplasma pneumoniae infection was obtained in ten patients (29%), eight of whom were found positive by both PCR and serology. Two of the sputum samples from these eight patients were negative by culture. Of the remaining two patients positive for Mycoplasma pneumoniae, one was positive by PCR and culture but negative by serology, and one was found positive by serology but negative by PCR and culture. Thirteen of the 14 controls were negative by both PCR and serology. One control, however, was negative by serology but positive by PCR, which was probably due to asymptomatic carriage of Mycoplasma pneumoniae. The results of this study indicate the suitability of the PCR for the detection of Mycoplasma pneumoniae in clinical samples as well as its potential value as an additional tool for the diagnosis of infection.     

Changes in the microflora of the sputum and the bronchoalveolar fluid in patients with acute and protracted pneumonias

A quantitative technique was used to microbiological study of sputum in 110 patients with acute pneumonia and 56 with advanced pneumonia, in 38 of them, their bronchoalveolar lavage fluid was simultaneously examined. In acute pneumonia, Pneumococcus was most commonly (71.8%) cultured, frequently in combination with other microorganisms, mainly with Neisseria and Enterobacteriaceae. These patients were found to have higher pneumococcal cultivation rates (83.0%) in the bronchoalveolar lavage fluid. Following 3 weeks of etiotropic therapy, the pneumococcal cultivation rates dropped to 10.8%. Pneumococcus also occupied the leading place (69.6%) in the etiology of advanced pneumonia, Mycoplasma pneumoniae was detected by the indirect immunofluorescence test in 28.5% of patients. After 3-week therapy, the cultivation rates for Pneumococcus decreased to 23.0%, its association with other microbes being more frequently observed. At the same time there was a rise in the detection rate of Mycoplasma antigen, which could cause advanced pneumonia.     

Heparin therapy: 1998 (current use of the 80-year-old heparin)

OBJECTIVES: Describe the different features of a common disease: Mycoplasma pneumoniae pneumonia. PATIENTS AND METHODS: The hospital files of 10 consecutive patients with microbiologically proven Mycoplasma pneumoniae pneumonia were reviewed retrospectively. These 10 patients were hospitalized over a 15-month period among 150 patients admitted to the Versailles general hospital for community-acquired pneumonia. We compared our series with data in the literature. RESULTS: Most of the patients with Mycoplasma pneumoniae pneumonia were young apparently healthy adults. A bronchial risk factor (smoking, allergy) was however found in 60% of the patients. The principle symptom was persistent cough (100%), with fever and joint pain, or sometimes headache and signs of ENT involvement. Dyspnea was frequent, related more to associated bronchospasticity than to the severity of the pneumonia. Radiographic findings were quite variable. In one case hemolytic anemia and cold agglutinins suggested the diagnosis. Certain diagnosis was based on positive serology after hospitalization due to the long delay between symptom onset and hospitalization. The prehospital period was characterized by a succession of ineffective empirical antibiotic regimens. In routine practice, macrolides or fluoroquinolones administered for 2 to 3 weeks are the empirical antibiotics of choice. Outcome is generally favorable with rapid clinical and radiological improvement. Antibiotic therapy is not however sufficient alone to achieve improvement in the respiratory impairment: bronchodilators and corticosteroids are necessary to treat the bronchospasticity. CONCLUSION: Despite the benign nature of community-acquired pneumonia due to Mycoplasma pneumoniae, clinical manifestations, particularly bronchial inflammation may have important consequences.     

Comparison of two serological methods and a polymerase chain reaction-enzyme immunoassay for the diagnosis of acute respiratory infections with Chlamydia pneumoniae in adults

Chlamydia pneumoniae is a common respiratory tract pathogen. Serological methods currently used for the diagnosis of C. pneumoniae infection lack specificity, give ambiguous results from a single serum sample and often provide only a retrospective diagnosis. A prospective study was undertaken to assess whether PCR could be a useful addition to the serological techniques routinely practised for diagnosis. This study investigated 68 adult patients with a diagnosis of acute respiratory infection. Acute and convalescent serological determination of antibodies to C. pneumoniae were performed by means of an rELISA test and a micro-immunofluorescence (MIF) test. Nasopharyngeal aspirates or bronchoalveolar lavage specimens and bronchial aspirates obtained from the 68 patients were evaluated by PCR-enzyme immunoassay (PCR-EIA) for the presence of C. pneumoniae and by immunofluorescence assay and cell culture for virus identification. Mycoplasma pneumoniae serology was also performed. Eight patients (11.8%) were positive by either rELISA or PCR-EIA, or both, with an infection rate of 5 (18.5%) of 27 in patients with community-acquired pneumonia, 2 (9%) of 22 in asthmatic patients and 1 (5%) of 19 in patients with an exacerbation of chronic obstructive pulmonary disease. Serological evidence of acute infection was found in four of these patients with the rELISA test and in three others with the MIF test. PCR-EIA detected C. pneumoniae DNA in four specimens, but there were concordant results with both rELISA and PCR-EIA in only one patient A positive PCR-EIA was also obtained in a patient who did not show an antibody response in acute serum. The discrepancy between serological and PCR-EIA results reflects the difficulties in routine laboratory diagnosis of C. pneumoniae infection and the necessity for further studies with optimised techniques.     

Community-acquired pneumonia: prospective study of 101 adult, immunocompetent patients for 1 year

BACKGROUND: A one year prospective study was carried out to assess the etiology of community-acquired pneumonia (CAP), and also to know the incidence, characteristics and evolution of infection by Chlamydia pneumoniae; and the effectiveness of DNA probes in CAP due to Mycoplasma pneumoniae and Legionella. METHODS: One hundred and ten patients with a diagnosis of CAP in the emergency department were studied. Serologic studies were performed, and also tests commonly used for the diagnosis of respiratory tract pathogens in respiratory samples, including serology and culture of Chlamydia pneumoniae and DNA probes for Mycoplasma pneumoniae and Legionella. RESULTS: In 72 cases (71.3%) some pathogen was found and in 5 cases more than one microorganism was involved. The etiology was bacterial in 31% of the cases, with S. pneumoniae being the most frequent (19 cases). Forty percent of the cases were "atypical" pneumonias with 33 cases of M. pneumoniae and 5 by Chlamydia pneumoniae. Diagnostic data of viral pneumonia were found in 2 cases. DNA probes were not useful for the diagnosis of pneumonia by Legionella pneumophila and had low effectiveness (31.8%) in Mycoplasma pneumoniae CAP. CONCLUSIONS: a) M. pneumoniae was the most frequent pathogen (33%). b) DNA probes for M. pneumoniae had low sensitivity in sputum (31.8%) and none in pharyngeal exudate. c) Acute infection by C. pneumoniae was diagnosed in 5 cases. Previous data of infection were recorded in 60.4% of the patients. d) Bacterial pneumonia (31%) was underestimated due to a low rate of bacteremic cases (7.9%) and the low number of positive cultures with definitive diagnostic value. e) The evolution was good except in two cases (death due to staphylococcal pneumonia with alcohol withdrawal syndrome and multiorganic failure by disseminated chicken-pox).     

A three-stage scheme for medical negligence

An eighteen-month prospective study designed to determine the incidence, etiology and prognosis of community acquired pneumonia (CAP) in adults requiring admission to hospital. METHODS: We studied 366 patients admitted to hospital after being diagnosed of CAP at the Emergency Room of a General Hospital. Standard laboratory methods were used for culture from blood and sputum, and serology tests for Legionella pneumophila. Mycoplasma pneumoniae, Chlamydia psittaci and Coxiella burnetti. Patients were evaluated until complete recovery, paying special attention to prognostic factors predictive of death. RESULTS: An etiological diagnosis was established in 99 patients (27.6%). Legionella pneumophila was the most common pathogen accounting for 30 cases (8.2%), followed by Streptococcus pneumoniae with 26 cases. 26 patients died (mortality rate of 7%); factors predictive of death included pre-existing disease, tachypnea and elevated blood urea nitrogen level. CONCLUSIONS: CAP represented 4.4% of admissions. Legionella pneumophila was the most frequently identified pathogen. If tachypnea and/or uremia are noted on admission, there is an increase in the risk of death.     

Experimentally induced Mycoplasma pneumoniae pneumonia in chimpanzees

Eight chimpanzees were examined. Two served as negative control and six inoculated with Mycoplasma pneumoniae became colonized. Colonization persisted for 28-68, 16-50 and 21 days with an average duration of 47, 32.5 and 21 days in the oropharyngeal, tracheal and lung tissues, respectively. Mycoplasma titers ranged from 10(8) to 10(1) color-changing units per specimen during the course of the infections. Seroconversion occurred within 12-15 days and peak antibody titers ranged from 1.256 to 1.1024 and developed between days 28 and 48 post-inoculation. Positive cold agglutinin titers were detected between 12 to 15 days and peak titers ranged from 1:80 to 1:640. Significant increases in sIgA and IgG immunoglobulin antibody levels were detected in lung lavage fluids. Unlike the many other experimentally infected animals examined, chimpanzees infected with M. pneumoniae had positive X-ray findings, developed cold agglutinins and showed overt signs of disease. These signs include persistent cough, low grade fever, rhinitis, oropharyngitis, diarrhea, and loss of appetite. Peak severity of disease corresponded with peak lung colonization, and the detection of cold agglutinins and positive X-ray findings. The microbiological, serological and clinical aspects of pneumonia induced in chimpanzees was similar to naturally occurring primary atypical pneumonia in humans.     

Safety and efficacy of azithromycin in the treatment of community-acquired pneumonia in children

OBJECTIVE: To compare the safety and efficacy of azithromycin with amoxicillin/clavulanate or erythromycin for the treatment of community-acquired pneumonia, including atypical pneumonia caused by Mycoplasma pneumoniae and Chlamydia pneumoniae. METHODS: Multicenter, parallel group, double blind trial in which patients 6 months to 16 years of age with community-acquired pneumonia were randomized 2:1 to receive either azithromycin for 5 days or conventional therapy for 10 days (amoxicillin/clavulanate if < or =5 years of age or erythromycin estolate if >5 years of age). Patients from 23 geographically diverse sites were evaluated for clinical outcomes and/or adverse events at Days 3 to 5, Days 15 to 19 and 4 to 6 weeks posttherapy. Microbiology (culture or polymerase chain reaction) was done at baseline and Days 15 to 19 for bacteria, Chlamydia pneumoniae and Mycoplasma pneumoniae. Serology for C. pneumoniae and M. pneumoniae was done at baseline and 4 to 6 weeks posttherapy. RESULTS: Of 456 patients enrolled during 17 consecutive months, 420 were evaluable. Clinical success at Study Days 15 to 19 was 94.6% in the azithromycin group and 96.2% in the comparative treatment group (P = 0.735) and at 4 to 6 weeks posttherapy 90.6 and 87.1%, respectively (P = 0.330). Evidence of infection was identified in 46% of 420 evaluable patients (1.9% bacteria, 29.5% M. pneumoniae and 15% C. pneumoniae). Microbiologic eradication was 81% for C. pneumoniae and 100% for M. pneumoniae in the azithromycin group vs. 100 and 57%, respectively, in the comparator group. Treatment-related adverse events occurred in 11.3% of the azithromycin group and 31% in the comparator group (P < 0.05). CONCLUSION: Azithromycin used once daily for 5 days produced a satisfactory therapeutic outcome similar to those of amoxicillin/clavulanate or erythromycin given three times a day for 10 days for treatment of community-acquired pneumonia. Azithromycin had significantly fewer side effects than comparator drugs.     

Diagnosis of Mycoplasma pneumoniae pneumonia in pediatric patients by polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) testing was performed on respiratory tract specimens obtained by throat swab in 21 children admitted to the hospital with suspected Mycoplasma pneumoniae pneumonia. Of 13 patients with a clinical condition compatible with mycoplasma infection and an immunological response to M. pneumoniae, 11 were positive by PCR. Eight patients were negative by serology and/or had a clinical condition not compatible with mycoplasma infection, and all were negative by PCR. The antibody response to M. pneumoniae was delayed for a week or more in 3 (23%) of the 13 patients with documented mycoplasma infection. These results suggest that PCR performed on a respiratory tract specimen obtained by a throat swab may be useful in the initial evaluation of children with suspected M. pneumoniae pneumonia, especially in patients in whom the serological response is delayed.     

The tumor-suppressor function of E-cadherin

STUDY OBJECTIVE: To compare the etiology of community-acquired pneumonia in Japan and Western countries, the causative pathogens were prospectively investigated in patients requiring hospitalization. DESIGN: Prospective study over a 3-year period. SETTING: A community general hospital in Japan. PATIENTS: Three hundred twenty-six episodes of community-acquired pneumonia in 318 patients admitted to the hospital between July 1994 and June 1997. METHODS: The microbiological diagnosis was based on the results of quantitative sputum culture, blood culture, and other invasive procedures, including transthoracic needle aspiration or bronchoscopic examination. Serologic tests for Mycoplasma pneumoniae, Chlamydia spp, Legionella spp, and viruses were also routinely performed. RESULTS: Causative pathogens were identified in 199 episodes (61%). Streptococcus pneumoniae was the most common pathogen (23%), followed by Haemophilus influenzae (7.4%), M pneumoniae (4.9%), and Klebsiella pneumoniae (4.3%). The Streptococcus milleri group and Chlamydia pneumoniae were detected in 3.7 and 3.4% of the episodes, respectively. Pneumonia due to Legionella spp was recognized in only two patients. CONCLUSIONS: The etiology of community-acquired pneumonia in Japan did not differ markedly when compared with that of Western countries except for the low incidence of Legionella pneumonia. C pneumoniae and the S milleri group, which are emerging or newly recognized pathogens, were also significant causative microorganisms.     

Etiology of childhood pneumonia: serologic results of a prospective, population-based study

BACKGROUND: To investigate the etiology of pediatric community-acquired pneumonia, we conducted a prospective, population-based study covering the total population <15 years of age (n = 8851) in 4 municipalities in eastern Finland. MATERIALS AND METHODS: The number of patients was 201; chest radiographs were available for all cases and paired sera for serologic assays were available for >90% of cases. The methods included assays for antibody response to 3 pneumococcal antigens, specific pneumococcal immune complex assays and conventional antibody tests for mycoplasmal, chlamydial and viral infections. RESULTS: Serologic evidence of specific microbial etiology was obtained in 133 (66%) of the pneumonia patients. Bacterial infection was diagnosed in 102 cases (51%) and viral infection in 51 cases (25%). Streptococcus pneumoniae was the most common agent (57 cases; 28%), followed by Mycoplasma pneumoniae (44; 22%), respiratory syncytial virus (43; 21%) and Chlamydia spp. (29; 14%). Haemophilus influenzae was identified in only 6% and Moraxella catarrhalis in only 3% of the children. More than one specific infection was found in 51 patients (25%). The proportion of pneumococcal cases varied from 24 to 36% by age. Mycoplasma infections were seen mostly in patients > or =5 years and Chlamydia infections in patients > or =10 years of age. CONCLUSIONS: The results of our prospective, strictly population-based study confirm the importance of S. pneumoniae in the etiology of community-acquired pneumonia in children of all ages. M. pneumoniae and Chlamydia pneumoniae are important from the age of 5 years onwards.     

Epidemiology of acute bronchopulmonary infections in children

In infants and young children acute lower respiratory infection is the most common cause of morbidity and death especially in developing countries. Factors that contribute to the increased susceptibility to respiratory pathogens include young age, season, sex, indoor pollution, large family size, malnutrition, low immunocompetence, socioeconomic disadvantage. The epidemiology of acute respiratory infections in childhood seems similar worldwide. In all countries, respiratory syncytial virus, parainfluenzae virus 1 and 3 influenzae A and B viruses and adenovirus are reported to be the main causes of acute respiratory infections. Six microorganisms are responsible of 90% of documented acute bacterial pulmonary infections, Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydia pneumoniae, Chlamydia trachomatis, Haemophilus influenzae, Staphylococcus. Mixed viral and bacterial infections occur frequently (30%). The role of respiratory viruses in predisposing to colonization and invasion of bacterial organisms has often been suggested. In recent years acquired resistance against antibiotic for H. influenzae and S. pneumoniae has emerged.     

Diagnosis of mycoplasma infections using directed amplification

Test systems for rapid detection of mycoplasmas in biological samples have been elaborated on the base of the polymerase chain reaction (PCR). Amplification of the conservative rDNA sequences was used for testing of cell cultures for mycoplasmal contamination. Mycoplasma pneumoniae detection system has been developed based on amplification of the species-specific DNA sequences. Inversions of some repeated sequences in the Mycoplasma pneumoniae genome make it possible to run the PCR with a single primer. The revealed spacer length polymorphism for 16S-23S rDNA operons can be used in the mycoplasmas identification.     

Septic arthritis caused by Streptococcus pneumoniae

BACKGROUND: Streptococcus pneumoniae is an uncommon agent of infective arthritis. In this report three cases of pneumococcal arthritis are described. METHODS: Retrospective review of synovial fluids processed in our laboratory yielding bacteria. The study period was from January 1991 to December 1995. The clinical records of patients with the clinical and microbiological diagnosis of septic arthritis were reviewed. RESULTS: Twenty-eight out of a total of 43 clinical records had the clinical and microbiological diagnosis of septic arthritis and three (11%) were caused by Streptococcus pneumoniae. The infective source in two of these three cases was probably the respiratory tract, and the most common location was the knee. CONCLUSIONS: In our cases immunosuppression seemed to be the major risk factor involved in the development of pneumococcal arthritis.     

Long-term results after repeated surgical removal of pulmonary metastases

A 14-year-old boy developed acute transverse myelitis with severe abdominal pain, bladder dysfunction, weakness, and sensory loss of the lower extremities. Magnetic resonance imaging revealed a segmental expanded central edema affecting parts of the spinal cord, including the caudal medulla oblongata. Antibody response to Mycoplasma pneumoniae was negative in microparticle agglutination assays (1:40 in the acute serum and 1:160 in the convalescent serum) and complement fixation tests (1:20 and 1:10). However, analysis of acute-phase serum revealed a specific IgA and IgG response but no IgM response. Detection of M. pneumoniae in the cerebrospinal fluid by nested polymerase chain reaction and in nasopharyngeal aspirate by culture confirmed an M. pneumoniae infection. Treatment with doxycycline (100 mg daily) was started on the second day after admission to the hospital and continued for 14 days; the patient recovered completely and was discharged 20 days after onset of the disease, with no signs of neurological deficits.     

Transfusion management of an IgA deficient patient with anti-IgA and incidental correction of IgA deficiency after allogeneic bone marrow transplantation

In serology, lack of specificity can generally be attributed to cross-reactions between different pathogens with antigens bearing similar epitopes. During seroepidemiologic surveys of contagious agalactia of sheep caused by Mycoplasma agalactiae infection, numerous sera were analyzed by enzyme-linked immunosorbent assay (ELISA). A few sera reacted with various antigens coated on plates, including the well with no antigen. This reactivity was not due to cross-reactions as initially suspected, and these multipositive sera were designated false-positive sera. Elimination of this false positivity was not possible by using covalent ELISA plates or different rabbit anti-sheep IgG conjugates. Only conjugates using monoclonal antibodies or protein G were efficient in elimination of false positivities without reducing the true specific positive titers. No false-positive sera have been observed since the implementation of protein G conjugates in the serologic diagnosis of contagious agalactia by ELISA for the past 2 years.     

Dens evaginatus on a wisdom tooth: a diagnostic dilemma. Case report

BACKGROUND: Skin manifestations have been described in 25% of patients with Mycoplasma pneumoniae infection. CASE REPORT: We report a case of Mycoplasma pneumoniae infection in a 29-year-old man who developed a polymorphous erythema-like reaction. Skin manifestations were associated with voluminous lymph node enlargement and high eosinophil levels both in serum and alveolar lavage. Seroconversion against Mycoplasma pneumoniae IgG was documented with ELISA. The clinical course was favorable with erythromycin. DISCUSSION: ELISA IgG seroconversion is sufficient to confirm the diagnosis of Mycoplasma pneumoniae infection as this test has an 80-90% specificity. This case was unusual by its clinical presentation and high eosinophil counts in serum and tissue samples, similar to what is found in drug-induced hypersensitivity.     

Molecular biology of Mycoplasma

Mycoplasmas are the smallest free living microorganisms with the smallest genome. The G+C content is in general low (25-33%) and the coding capacity is about 600 proteins. Mycoplasma species are phylogenetically related, they use the genetic codon UGA for tryptophan, and show rapid evolution, with a high rate of divergence. The genomes of Mycoplasma genitalium and Mycoplasma pneumoniae have been fully sequenced. Striking features of the M. genitalium sequencing project are the presence of a high number of membrane proteins with no resemblance to previously sequenced genes and the presence of repeated fragments of the gene encoding the tip-localized 140 kDa adhesin (MgPa). Many Mycoplasma species display a high frequency of antigenic variation, both as phase and size variation of individual antigens. Mycoplasma hominis isolates are known to be antigenic heterogeneous, as reflected in the reactivity with monoclonal antibodies (MAbs). The genetics of the antigenic variation has been studied for three different surface exposed antigens: P120, Lmp, and P50/Vaa. The gene encoding P120 had a hyper-variable region in the N-terminal region. In addition, a second gene with homology to p120 was identified. The gene encoding Lmp, a 135 kDa protein is repeated and both genes are translated and both contain internal repeated sequences. Deletion mutants in the lmp gene were obtained by cultivation of M. hominis PG21 with MAb 552 specific for the repeated part of Lmp. One of the lmp genes had deletions of from four to eight repeats. The other gene was left unaltered. The genes encoding P50/Vaa show a different form of variability where domains of the genes seem to be exchangeable. The genomic maps of five M. hominis strains showed that even though the size of the genomes varied the position of the different genes were in general conserved.     

Detection of Mycobacterium tuberculosis DNA in lobular granulomatous panniculitis (erythema induratum-nodular vasculitis)

The antimicrobial spectrum of azithromycin and clarithromycin suggests a number of further uses for these newer macrolides. Favorable clinical and bacteriologic responses have been reported with both antibiotics in children with community-acquired pneumonia. Response rates were high for overall patient populations and for subgroups with infection caused by Mycoplasma pneumoniae and Chlamydia pneumoniae. Treatment with azithromycin or clarithromycin has resulted in a reduction in mycobacteremia and an improvement in clinical symptoms in adult AIDS patients with disseminated Mycobacterium avium-intracellulare complex. Prophylactic treatment with azithromycin may prevent M. avium-intracellulare complex, especially when combined with rifabutin. Preliminary evidence suggests that both azithromycin and clarithromycin in multidrug combinations may effectively eradicate Helicobacter pylori and that azithromycin may be useful in treating bacterial gastritis caused by Campylobacter species. Trachoma and infections caused by Bordetella pertussis and Ureaplasma urealyticum are other possible future indications for the newer macrolides. Limited clinical evidence also suggests that azithromycin may be effective in the prevention and treatment of malaria.     

Acute pancreatitis in Mycoplasma pneumoniae infections

Report on a man of 29 years with acute bronchopneumonia, antibodies against Mycoplasma pneumoniae and acute pancreatitis lasting three weeks. In some cases M. p. must be considered to be the cause of acute pancreatitis.