Detection of metabolic inhibition antibody to Mycoplasma pneumoniae by new method with guinea-pig complement

Improvement of the fermentation-inhibition (FI) test for Mycoplasma pneumoniae was attempted. The sensitivity of detecting the FI antibody to M. pneumoniae in the homologous immune rabbit serum was notably elevated, when such FI medium containing gamma globulin-free horse serum and guinea-pig complement in substitution for unheated horse serum was used. The M. pneumoniae suspension was filtered to remove aggregates of the organisms and used as the antigen for this new FI test. In detection of the serum antibody of patients with M. pneumoniae infections, the new FI test showed much higher sensitivity than conventional FI test and strongly correlated (r = 0.84) with high density particle agglutination test known to detect both IgG and IgM antibodies.     

A simple and sensitive method for detecting bacterial elastase production

A sensitive method for detecting bacterial elastase production in growing cultures is described. A variety of commonly isolated clinically relevant aerobic and anaerobic bacteria have been shown to produce the enzyme.     

Non-specific polyclonal antibody response induced by Mycoplasma pneumoniae

The ability of heat-killed Mycoplasma pneumoniae (MP) organisms to induce polyclonal antibody production in cultures of blood lymphocytes of healthy subjects was studied. MP induced both IgM and IgG production, with a predominance of IgM. Supernatants of MP-stimulated lymphocyte cultures were tested by an enzyme-linked immunosorbent assay for antibodies to measles, rubella, and herpes simplex virus. MP as well as pokeweed mitogen induced production of viral antibodies of IgG class in lymphocytes of donors who had serum antibodies to the corresponding viral antigens. The MP-induced non-specific antibody response was T-cell-dependent. Lymphocytes from four patients with MP pneumonia, collected nine to 13 days after onset of illness, were tested for in vitro Ig production in the absence of MP. These lymphocytes spontaneously produced increased amounts of IgM and/or IgG. Lymphocytes from three of these four patients spontaneously produced viral IgG antibodies to measles and/or varicella antigens, indicating that MP had induced non-specific activation of memory B cells in vivo. Spontaneous viral antibody production was not found in lymphocyte cultures of healthy donors. The non-specific activation of blood B cells in vitro is probably induced by non-specific helper factors from MP-activated T cells. It is possible that in vivo MP also may have a direct activating effect on B cells.     

A fast and sensitive method for detecting specific viral RNA in mammalian cells.

A quick and sensitive method to quantitate viral RNA synthesis has been developed. Utilizing glutaraldehyde to fix infected cells onto nitrocellulose paper, viral RNA can be probed directly in situ. Viral message can be detected from as few as 10(4) infected cells. This technique can be used to study viral gene expression and can be adapted to screen the activity of antiviral agents such as interferon.     

A sensitive method for detecting proteolytic enzymes released from their supports

Biotechnology Letters - A very sensitive radioisotope method, using 125I-labelled serum albumin as substrate, is proposed for the detection of very low enzymatic activities released from insoluble...     

Purification of a Mycoplasma pneumoniae adhesin by monoclonal antibody affinity chromatography.

A 165,000-dalton surface protein of Mycoplasma pneumoniae, designated protein P1, appears to be the major attachment ligand of the pathogen. We employed monoclonal antibody affinity chromatography to obtain purified protein P1.     

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.     

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.     

The creation of a test system for detecting Mycoplasma pneumoniae based on a method of directed DNA amplification]

A system for the rapid detection of M. pneumoniae, the causative agent of pneumonia in humans, has been developed. This system is based on the amplification of M. pneumoniae chromosomal DNA sequences in the polymerase chain reaction (PCR). The use of two primer sets, for nucleotide sequences of adhesion protein P1 and for nucleotide sequences of variable regions of the 16S ribosomal RNA, has permitted the detection of individual M. pneumoniae cells. The application of this technique for the study of simulated clinical material has shown that PRC is a sensitive and reliable assay and may be useful for the early detection of M. pneumoniae in infectious clinical material (blood and sputum samples).     

Motility of Mycoplasma pneumoniae.

Cell of Mycoplasma pneumoniae FH gliding on a glass surface in liquid medium were examined by microscopic observation and quantitatively by microcinematography (30 frames per min). Comparisons were made only within the individual experiments. The cells moved in an irregular pattern with numerous narrow bends and circles. They never changed their leading end. The average speed (without pauses) was relatively constant between o.2 and 0.5 mum/s. The maximum speed was about 1.5 to 2.0 mum/s. The movements were interrupted by resting periods of different lengths and frequency. Temperature, viscosity, pH, and the presence of yeast extract in the medium influenced the motility significantly; changes in glucose, calcium ions, and serum content were less effective. The movements were affected by iodoacetate, p-mercuribenzoate, and mitomycin C at inhibitory or subinhibitory concentrations. Sodium fluoride, sodium cyanide, dinitrophenol, chloramphenicol, puromycin, cholchicin, and cytochalasin B at minimal inhibitory concentrations did not affect motility. The movements were effectively inhibited by anti-M. pneumoniae antiserum. Studies with absorbed antiserum suggested that the surface components involved in motility are heat labile. The gliding of M. pneumoniae cells required an intact energy metabolism and the proteins involved seemed to have a low turnover.     

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.