Single tube real time PCR for detection of Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila from clinical samples of CAP

We designed a multiplex real time PCR for rapid, sensitive and specific detection of Streptococcus pneumoniae, Legionella pneumophila, Chlamydophila pneumoniae and Mycoplasma pneumoniae. The study cases consisted of 129 patients with community acquired pneumonia (CAP). Bacteriological techniques were implemented for detection of the cultivable organisms. DNA were extracted from sputa, throat swabs, bronchoalveolar lavages and tracheal aspirates and used as templates in real time PCR. The primers and probes were designed for cbpA (S. pneumoniae), p1adhesin (M. pneumoniae), mip (L. pneumophila) and ompA (C. pneumoniae). After optimization of real time PCR for every organism, the experiments were continued in multiplex in a single tube. Of 129 CAP specimens, the positive cultures included 14 (10.85%) for S. pneumoniae, 9 (6.98%) for L. pneumophila and 3 (2.33%) for M. pneumoniae. Four specimens (3.10%) were positive for C. pneumoniae by real time PCR. The sensitivity of our real time PCR was 100% for all selected bacteria. The specificity of the test was 98.26%, 98.34%, 100% and 100% for S. pneumoniae, L. pneumophila, M. pneumoniae and C. pneumoniae, respectively. This is the first report on the use of multiplex real time PCR for detection of CAP patients in the Middle East. The method covers more than 90% of the bacterial pathogens causing CAP.     

Polymerase chain reaction as a sensitive and rapid method for specific detection of Mycoplasma pneumoniae in clinical samples

The fast diagnosis of Mycoplasma primary atypical pneumonia is impaired by the lack of routinely available culture methods for isolation of Mycoplasma pneumoniae from clinical specimens. Likewise, serological methods commonly used for diagnosis are insensitive and non-specific. In this study, we have established and applied the polymerase chain reaction (PCR) technique to detect M. pneumoniae DNA in clinical samples originating from the respiratory tract. The PCR results were compared with those from culture and serology tests. To standardize the detection of M. pneumoniae by PCR, we first used DNA from culture grown organisms and clinical samples seeded with M. pneumoniae. PCR amplification was performed with M. pneumoniae-specific primers to amplify 144, 153 and 631 bp DNA fragments by using primer pairs MP5-1/MP5-2, P1-178/P1-331 and P1-178/P1-809, respectively. The amplification of the 631 bp DNA fragment was found to be most sensitive for the detection of M. pneumoniae. Using the most sensitive PCR, a total of 47 respiratory specimens from patients suspected of community acquired pneumonia were tested. While none of the specimens were positive for M. pneumoniae in culture, 6 specimens gave positive results by PCR. In 4 out of the 5 PCR positive samples tested serologically, the results were supported by elevated levels of anti-mycoplasma IgG/IgM/IgA. Thus, these results suggest that PCR is the most sensitive method to detect M. pneumoniae in clinical specimens.     

M. pneumoniae respiratory diseases: clinical features--children

Chest X-ray findings were studied in 618 pediatric patients with M. pneumoniae respiratory infections. Of these, 472 (76 percent) had pneumonia. Pneumonia was most frequently observed in the lower lung field and least frequently in the upper lung field. The enlargement of hilar lymph nodes was observed in 34 percent of patients with M. pneumoniae pneumonia in contrast to 5 to 9 percent of patients with pneumonia due to other agents, suggesting that it was rather characteristic of M. pneumoniae pneumonia. It was observed in no patients below one year of age, in 41 percent of those aged one to five years, and then decreased with increase in age. Of children with M. pneumoniae respiratory infections, fever, pneumonia, and positive CF test were less frequently observed in infants below one year, showing that they have slighter symptoms; positive IHA test was less frequently observed and isolation of M. pneumoniae was more frequently observed, as compared to other age groups, among whom these findings were similar. It must be kept in mind, however, that fatal cases of M. pneumoniae pneumonia in infants were reported.     

Evaluation of the NucliSens miniMAG RNA extraction and real-time NASBA applications for the detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae in throat swabs

Nucleic acids were extracted from 215 throat swabs from patients with community-acquired pneumonia by the manual Boom extraction, the NucliSens miniMAG and the Qiagen DNA blood kit and amplified respectively by in-house real-time NASBA, NucliSens EasyQ reagents, and real-time PCR for the detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae. Five out of 215 throat swabs were found to be C. pneumoniae positive by all techniques used. A total of 11 out of 215 throat swabs were positive for M. pneumoniae; 10/215 by Qiagen extraction and PCR amplification and 9/215 by NucliSens miniMAG and NucliSens EasyQ amplification. The NucliSens miniMAG and NucliSens EasyQ applications were successfully coupled to detect both organisms in throat swabs.     

Prevalence of Mycoplasma pneumoniae and Chlamydia pneumoniae in pneumonia patients in four major hospitals in Trinidad

The prevalence of current Mycoplasma pneumoniae and Chlamydia pneumoniae infections in patients with pneumonia in Trinidad, and the relationship between pneumonia and risk factors were investigated. Blood samples were collected from 132 patients diagnosed by attending physicians, as suffering from pneumonia at four hospitals in Trinidad. Serum samples were tested for M. pneumoniae IgM and IgG and C. pneumoniae IgM by the enzyme immunoassay (EIA). In addition, C. pneumoniae IgM and IgG were detected using microimmunofluorescence (MIF). A comprehensive questionnaire which addressed demographic information as well as risk factors for pneumonia was administered to patients. All analyses were done using the Statistical Package for Social Sciences (SPSS), version 9. Seroprevalences of 46.0% (58 of 126) were found for C. pneumoniae Ig M/G, and 66.7% (88 of 132) for M. pneumoniae Ig M/G. The difference was statistically significant (p < 0.01; chi2). Thirty-four percent (43 of 125) for C. pneumoniae Ig M/acute Ig G and 28.8% (36 of 125) of M. pneumoniae IgM were not statistically significant (p > 0.05; chi2). Hospital, gender and ethnicity of patients did not significantly (p > 0.05; chi2) affect the seroprevalence of the bacteria assayed for. However, the prevalence of C. pneumoniae (23.3%) in patients under 21 years old compared to other age groups was statistically significant (p = 0.043; chi2). Overall, the seroprevalence to both pathogens was not significantly (p > 0.05; chi2) affected by comorbidities and signs/symptoms. It was concluded that new infections by C. pneumoniae in pneumonia patients may be an important aetiological agent for the condition in Trinidad.     

Lobar pneumonia caused by Mycoplasma pneumoniae.

Seven patients with Mycoplasma pneumoniae pneumonia presented with moderate to dense consolidation in one (in five patients) or more lobes. The diagnosis was suspected in five patients after failure to respond to 1 to 6 (average 2.6) antibiotics administered for 2 to 12 (average 7) days, and in one patient upon the development of hemolytic anemia. Clues to the diagnosis of nonbacterial pneumonia included a nonrespiratory viral-like prodromal period (in five), a nonproductive cough (in five), lack of rigors (in seven), recent "pneumonia" in family members (in ;three), normal total leukocyte and neutrophil counts (in six) and the absence of bacterial pathogens in smears and cultures of sputum (in all seven). The diagnosis of M. pneumoniae infection was supported by the presence of cold agglutinins (in a titre of 1.64 or greater) in ;the serum of five or six patients and was confirmed by diagnostic levels or increases in the titre of M. pneumoniae complement fixing antibodies. Awareness of the fact that M. pneumoniae can present as lobar consolidation and close attention to the clinical and laboratory data can usually suggest a nonbacterial cause and thus prevent delay in appropriate antibiotic treatment.     

Development of a PCR-based method for diagnosing Mycoplasma pneumoniae infections

The polymerase chain reaction was used to detect clinical samples of Mycoplasma pneumoniae. A 245-bp region of the cytoadhesin P1 gene was shown to be specifically amplified in Myc. pneumoniae , but not in other species of Mollicutes. Picogram amounts of Myc. pneumoniae DNA could be detected per ml blood serum by use of a simple and reliable protocol for sample preparation and a PCR reaction involving two rounds of amplification. Application of the PCR-based method for the detection of Myc. pneumoniae in serum samples and throat swabs from patients with atypical pneumonia showed that it could be used in clinical diagnosis.     

Serum immunoglobulin in Mycoplasma pneumoniae infection

The dynamics and nature of serum antibodies in experimental and natural M. pneumoniae infection have been studied. The synthesis of specific IgM, IgA and IgG has been found to occur in the course of infection. During repeated M. pneumoniae infection in guinea pigs, as well as in cases of acute and chronic mycoplasmal pneumonia in humans (at the acute period of the disease), the prevalence of the synthesis of specific IgM is observed. At the acute period of the disease a rise in the quantitative levels of IgM and IgG occurs in patients. High titers of specific IgM (1:32 and greater) determined at the acute period of the disease can serve as the diagnostic criterion of the mycoplasmal etiology of pneumonia.     

Mycoplasma pneumoniae infections in a rural setting in Canada.

Mycoplasma pneumoniae infection was monitored in patients with symptoms of acute respiratory tract infection in a village in southeastern Ontario from April 1983 to April 1984. M. pneumoniae was isolated from 51 (48%) of the 106 patients. The incidence began to increase in May 1983, reached a peak in July and declined to normal by mid-August. During the epidemic period M. pneumoniae was detected in 36 of the 43 symptomatic patients. The most prominent features of the outbreak were the considerable intrafamilial attack rate and the high frequency of pneumonia among infected patients. Treatment with tetracyclines and erythromycin reduced the duration of the illness and accelerated the resolution of symptoms.     

Assessment of real-time PCR for diagnosis of Mycoplasma pneumoniae pneumonia in pediatric patients

We developed a real-time PCR to detect Mycoplasma pneumoniae with a primer set designed for the 16S rRNA gene. Clinical samples (n=937) were collected from children with community-acquired pneumonia between April 2002 and March 2004 at 12 Japanese medical institutions. Sensitivity of real-time PCR was calculated as 10 colony-forming units per reaction tube using a pMP01 plasmid carrying a 225-bp target DNA fragment of the 16S rRNA gene in M. pneumoniae M129, a standard strain. Results, obtained within 2 h, were compared with those of conventional culture and serologic methods. Of all cases tested, 151 (16.4%) and 129 (13.8%) were positive for M. pneumoniae by real-time PCR and by culture, respectively. Among the 151 cases, almost all of those tested serologically by passive agglutination showed a rise in M. pneumoniae antibody titre between acute and convalescent sera. We conclude that this real-time PCR can identify M. pneumoniae rapidly and fulfills the need for rapid identification, high sensitivity, and high specificity.     

Antibodies anti-Chlamydia pneumoniae and anti-Mycoplasma pneumoniae in patients with negative serology for hantavirus. Retrospective study.

The seroprevalence of Chlamydia pneumoniae and Mycoplasma pneumoniae in hantavirus seronegative patients, who had symptoms and signs compatible with pneumonia was established. For this purpose we used the indirect fluorescent antibody test. Titers > or = 1:16 for C. pneumoniae and M. pneumoniae were found in 8.6% and 17.1% of the serum, respectively, showing evidence of recent or current infection.     

Evaluation of real-time quantitative PCR for identification and quantification of Chlamydia pneumoniae by comparison with immunohistochemistry.

Chlamydia pneumoniae is a common cause of community-acquired pneumonia and it has been associated with atherosclerosis. C. pneumoniae has usually been diagnosed by serology using a microimmunofluorescence test, but more recently polymerase chain reaction (PCR) has been viewed as an advantageous alternative. We developed a quantitative real-time PCR for detection of C. pneumoniae. Primers were targeted for the pmp4 gene, and the PCR fragment was detected real-time with a fluorescence resonance energy transfer probe set using a LightCycler instrument. The PCR was used on DNA released from 50 microm sections of paraffin-embedded formalin-fixed lung tissue from experimentally infected mice. Thereby, the number of C. pneumoniae genomes was determined. To our knowledge this is the first time quantification of C. pneumoniae DNA has been attempted on paraffin-embedded formalin-fixed tissue. C. pneumoniae-specific immunohistochemistry (IHC) was done on 5 microm sections adjacent to the sections used in PCR, and the number of inclusions were counted in each section. Good correlation was found when comparing results from PCR and IHC, which is in contrast to many previous studies.     

Circulating immune complexes in the diagnosis of Mycoplasma pneumoniae infection

The possibility of detecting M. pneumoniae antigen and antibodies to it, incorporated into immune complexes, in the sera of patients with acute pneumonia by means of erythrocyte diagnosticums was studied, and the immunological characterization of these complexes was made. In patients with mycoplasmal pneumonia M. pneumoniae antigen and specific antibodies, both free and incorporated into immune complexes, were found to circulate in the blood. In children, antigenemia was detected twice as frequently as in adults. Dissociated M. pneumoniae antigens had different molecular weight, their location on the gel chromatogram of the serum being in fractions 7S and 19S. The dissociation of immune complexes permits the detection of M. pneumoniae antigen and antibodies to it in a bound state by means of the passive hemagglutination test, thus increasing the frequency of positive results in the diagnosis of M. pneumoniae infection.     

Urinary detection of Streptococcus pneumoniae antigen for diagnosis of pneumonia

Streptococcus pneumoniae is one of most common causes of community-acquired pneumonia. We evaluated a newly available rapid immunochromatographic test to detect S. pneumoniae in urine samples verifying its importance in the diagnosis of pneumococcal pneumonia. Our data, obtained from 104 patients with community-acquired pneumonia, show that Now S. pneumoniae Urinary Test is characterized by a sensitivity value of 77.7%, a specifity of 98.8%: positive and negative predictive values are 93.3% and 95.5%, respectively. In conclusion, Now S. pneumoniae Urinary Test should be a useful test to establish the etiology of community-acquired pneumonia.     

Detection of Mycoplasma pneumoniae in simulated and true clinical throat swab specimens by nanorod array-surface-enhanced Raman spectroscopy

The prokaryote Mycoplasma pneumoniae is a major cause of respiratory disease in humans, accounting for 20% of all community-acquired pneumonia and the leading cause of pneumonia in older children and young adults. The limitations of existing options for mycoplasma diagnosis highlight a critical need for a new detection platform with high sensitivity, specificity, and expediency. Here we evaluated silver nanorod arrays (NA) as a biosensing platform for detection and differentiation of M. pneumoniae in culture and in spiked and true clinical throat swab samples by surface-enhanced Raman spectroscopy (SERS). Three M. pneumoniae strains were reproducibly differentiated by NA-SERS with 95%-100% specificity and 94-100% sensitivity, and with a lower detection limit exceeding standard PCR. Analysis of throat swab samples spiked with M. pneumoniae yielded detection in a complex, clinically relevant background with >90% accuracy and high sensitivity. In addition, NA-SERS correctly classified with >97% accuracy, ten true clinical throat swab samples previously established by real-time PCR and culture to be positive or negative for M. pneumoniae. Our findings suggest that the unique biochemical specificity of Raman spectroscopy, combined with reproducible spectral enhancement by silver NA, holds great promise as a superior platform for rapid and sensitive detection and identification of M. pneumoniae, with potential for point-of-care application.     

Rapid detection of Mycoplasma pneumoniae in clinical samples by real-time PCR

M. pneumoniae is a common causative agent of community-acquired pneumonia in children. The diagnosis of such infections is usually based on serology using complement fixation or, more recently, enzyme-immuno assays. PCR has been shown to be a promising alternative. We have evaluated a real-time PCR assay targeting the P1 adhesion protein gene and compared it to a conventional semi-nested PCR assay with the 16S rDNA as target. Comparison of 147 specimens from 48 patients showed an overall agreement of 97.4%. Real-time PCR proved to be of equal value on clinical specimens as conventional PCR regarding sensitivity and specificity, but is clearly advantageous regarding speed, handling and number of samples that can be analyzed per run.     

肺炎链球菌去信号肽脂蛋白PsaA锌离子结合特性的初步表征

肺炎链球菌是细菌性肺炎的主要病原体。PsaA是各种肺炎链球菌共有的遗传保守的特异性表面金属结合脂蛋白。通过PCR扩增肺炎链球菌D39不含信号肽的PsaA基因片段,将其通过T4连接酶连接至含6His标签的表达载体PBAD/HisA中,转化表达宿主大肠杆菌Top-10后用L(+)-阿拉伯糖诱导重组蛋白的表达。重组蛋白经亲和镍柱纯化以后,用外切酶-重组肠激酶(REK)去除6His标签。感应偶合电浆质谱(ICP-MS)测得纯化的PsaA蛋白以1:1比例结合金属锌离子。进而,通过圆二色谱法分析金属离子的结合对蛋白二级结构中α-螺旋和β-片层含量的影响,荧光光谱研究蛋白结合锌离子的解离常数及结合当量,为进一步研究该蛋白在体外的金属结合特性及细菌的金属运输及毒力机制提供理论基础。     

Determination of Mycoplasma pneumoniae antigens and antibodies by the immunoenzyme method in patients with respiratory tract diseases

The optimum conditions for carrying out the enzyme immunoassay (EIA) with a view to determine M. pneumoniae antigen and antibodies to them in the sera of patients with different respiratory diseases were established. The use of the specially modified EIA technique made it possible to reveal that patients with tuberculosis and chronic pneumonia showed similar occurrence of M. pneumoniae (35.7% and 35.0% of cases, respectively), while in patients with pulmonary sarcoidosis M. pneumoniae occurred in 27.2% of cases. At the same time the occurrence of antibodies in patients with chronic pneumonia and sarcoidosis was more than three times greater than in tuberculosis patients.     

Fulminant Mycoplasma pneumoniae pneumonia.

The frequency of fulminant pneumonia due to Mycoplasma pneumoniae is relatively rare despite the high prevalence of Mycoplasma species infection in the general population. We recently encountered such a case and have reviewed the English-language literature on cases of M pneumoniae pneumonia that have resulted in respiratory failure or death. Due to host factors or on epidemiologic grounds, fulminant cases seem to be more common in young healthy adults, in males, and possibly in smokers among the 46 patients we found. An enhanced host cellular immune response may be responsible for the development of severe cases. A spectrum of small airways disease is characteristic, including cellular bronchiolitis and bronchiolitis obliterans with and without organizing pneumonia. Based largely on anecdotal experience, corticosteroid use may be salutary in patients with respiratory failure. For reasons that are not well known, the incidence of pulmonary thromboembolism is increased in fatal cases.     

Application of PCR ribotyping and tDNA-PCR for Klebsiella pneumoniae identification

PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping) and tRNA intergenic spacer (tDNA-PCR) were evaluated for their effectiveness in identification of clinical strains of Klebsiella pneumoniae and differentiation with related species. For this purpose both methods were applied to forty-three clinical isolates biochemically identified as K. pneumoniae subsp. pneumoniae isolated from patients clinical specimens attended at five hospitals in three Brazilian cities. References strains of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, K. oxytoca, K. planticola and Enterobacter aerogenes were also analyzed. Both PCR methods showed specific patterns for each species. A conserved PCR ribotype pattern was observed for all clinical K. pneumoniae isolates, while differing from other related analyzed species. tDNA-PCR revealed five distinct patterns among the K. pneumoniae clinical isolates studied, demonstrating a predominant group with 90.6% of isolates presenting the same pattern of K. pneumoniae type strain. Both PCR-based methods were not able to differentiate K. pneumoniae subspecies. On the basis of the results obtained, both methods were efficient to differentiate the Klebsiella species analyzed, as well as E. aerogenes. Meanwhile tDNA-PCR revealed different tRNA arrangements in K. pneumoniae, suggesting intra-species heterogeneity of their genome organization, the polymorphism of the intergenic spacers between 16S and 23S rRNA genes appears to be highly conserved whithin K. pneumoniae clinical isolates, showing that PCR ribotyping can be an useful tool for identification of K. pneumoniae isolates.