Evaluation of Serodia Myco II particle agglutination test for detecting Mycoplasma pneumoniae antibody: comparison with mu-capture ELISA and indirect immunofluorescence.

The Serodia Myco II particle agglutination test, which the manufacturers claim exclusively detects IgM antibody, was compared with two IgM-specific tests, a mu-capture ELISA, and indirect immunofluorescence for their ability to detect recent Mycoplasma pneumoniae infection. In general there was good agreement among the three tests, all three having similar sensitivity. One hundred and nine (78%) of serum samples gave concordant results in all three assays. Several sera gave positive particle agglutination titres, however, while being negative by the two other assays, and the Serodia Myco II test may not be as specific for detecting M pneumoniae IgM as the other two tests. While the Serodia Myco II test may be a good screening assay, it is unlikely to be a definitive test for M pneumoniae IgM, but may be better than the complement fixation test, particularly in younger patients in whom M pneumoniae IgM is found more frequently.     

Attachment of Mycoplasma pneumoniae to respiratory epithelium.

The attachment of radioisotope-labeled Mycoplasma pneumoniae to hamster tracheal rings in organ culture was examined by radioautography and liquid scintillation counting. Radioautographs of individual rings exposed for 8 h to (3H) thymidine-labeled virulent M. pneumoniae revealed a dense extracellular collection of emulsion grains along the luminal surface of epithelial cells. Similar exposure of rings to isotope-labeled avirulent M. pneumoniae resulted in no accumulation of emulsion grains. The numbers of attached virulent mycoplasmas, as measured by liquid scintillation counting of infected rings, were found to increase in a nearly linear fashion over an 8-h incubation period. Viability of the mycoplasmas and metabolic integrity of the tracheal rings were important for optimal attachment. Pretreatment of rings with neuraminidase or sodium periodate significantly impaired orgainism adherence. These data suggest a specificity of interation between virulent M. pneumoniae and tracheal epithelial cells that can be further examined through the use of isotopically labeled mycoplasmas.     

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.     

Application of indirect immunofluorescence to detection of Dientamoeba fragilis trophozoites in fecal specimens.

An indirect fluorescent-antibody (IFA) assay was carried out to examine for the presence of Dientamoeba fragilis trophozoites in preserved fecal specimens. Antiserum to D. fragilis trophozoites was raised in a rabbit with a dixenic culture of D. fragilis (ATCC 30948) from the American Type Culture Collection. After absorption with Klebsiella pneumoniae and Bacteroides vulgatus, the immune rabbit serum was used for examination by the IFA assay. A total of 155 clinical samples were tested; 42 with no parasites, 9 with D. fragilis, and 104 with other parasites. The IFA assay identified seven D. fragilis organisms. Two specimens with doubtful IFA assay readings showed very scanty amounts of D. fragilis trophozoites on stained smears. There were no false-positive IFA assay readings. The IFA assay appeared to be a promising method because of its speed in screening. The specificity of the IFA assay indicates that other diagnostic tests such as an enzyme-linked immunosorbent assay could be developed to identify D. fragilis antigens in fecal specimens.     

Comparison of direct and indirect immunofluorescence staining of clinical specimens for detection of respiratory syncytial virus antigen.

Immunofluorescence staining methods for respiratory syncytial virus antigen detection were compared. Of 50 specimens originally positive for respiratory syncytial virus by direct immunofluorescence and culture, 49 were positive by repeat direct immunofluorescence and 32 were positive by indirect immunofluorescence. Additional results obtained on specimens originally negative for respiratory syncytial virus by direct immunofluorescence, culture, or both indicate that direct immunofluorescence staining for respiratory syncytial virus antigen was more sensitive than was indirect immunofluorescence.     

DNA probes for detection and identification of Mycoplasma pneumoniae and Mycoplasma genitalium.

DNA probes specific for Mycoplasma pneumoniae and Mycoplasma genitalium were selected from genomic libraries prepared in pUC13. The 32P-labeled probes could detect, by dot blot hybridization, down to about 0.1 ng of the specific mycoplasma DNA or 10(5) CFU. Biotinylation of probe decreased the sensitivity of detection and produced nonspecific background reactions with nonhomologous DNAs. Sulfonation of probe yielded a similar level of sensitivity with less background.     

Multiplex polymerase chain reaction for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples.

AIMS: To develop a multiplex polymerase chain reaction (PCR) for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples. METHODS: Oligonucleotide primers for the amplification of the DNA of these three organisms were optimised for use in combination in the same reaction. PCR products were detected by hybridisation with pooled internal probes using an enzyme linked immunosorbent assay. Those with positive signals were further differentiated using species specific probes. Quality of DNA extraction and PCR inhibition were controlled by amplification of a human mitochondrial gene. A panel of 53 respiratory samples with known results was evaluated blindly. This was followed by a retrospective study on sputa collected from 244 patients with suspected community acquired pneumonia. RESULTS: The multiplex assay had a lower sensitivity than PCR with individual primers by about one log. The resultant sensitivity was considered acceptable for diagnostic use. Of the panel of 53 samples, nine of 11 M pneumoniae, 11 of 11 C pneumoniae, six of seven C psittaci, and 24 of 24 negative samples were correctly identified. Of the 244 patients with pneumonia, seven (2.9%) had detectable M pneumoniae, six (2.5%) had C pneumoniae, and one (0.4%) had C psittaci. The case notes from 11 patients were studied. The PCR finding was of possible significance in at least eight of these patients. CONCLUSIONS: This multiplex PCR assay has the potential to be used as a diagnostic and epidemiological tool. Further prospective studies are needed to establish its clinical value.     

The indirect platelet suspension immunofluorescence test in the detection of platelet antibodies in idiopathic thrombocytopenic purpura.

Among the various techniques developed for the detection of platelet antibodies, the platelet suspension immunofluorescence test has been reported to be simple, sensitive and reproducible, and therefore more clinically useful than other techniques available. An initial evaluation of the test was carried out for the detection of platelet autoantibodies in ten cases of idiopathic thrombocytopenic purpura. The indirect PSIFT was found to be positive in 60%. The technical aspects of the test and the problems encountered are discussed.     

Utility of an internal control for the polymerase chain reaction. Application to detection of Mycoplasma pneumoniae in clinical specimens.

The polymerase chain reaction (PCR) was used to amplify a 209 base-pair fragment of Mycoplasma pneumoniae DNA. The amplicon was transferred into a plasmid and a 680 base-pair piece of foreign DNA was inserted between the two amplimer sites. Plasmid DNA was added to the reaction mixture as an internal control for the polymerase chain reaction. Since the original hybridization target sites were included in this construction, one pair of amplimers could be used to amplify both the target DNA and the internal control DNA. Separation of internal control from target DNA after amplification was easily obtained on agarose gel electrophoresis. For the analysis of clinical samples with the polymerase chain reaction, the addition of internal control DNA allowed monitoring of the overall effectiveness of the amplification in each tube. With this technique approximately one-third of the tests were shown to be unsatisfactory due to technical errors or contaminating inhibitors. Adequate internal controls are necessary to avoid false-negative results with the polymerase chain reaction.     

Freeze-dried erythrocytes for an indirect hemagglutination test for detection of cytomegalovirus antibodies.

An indirect hemagglutination test with lyophilized, fixed, tanned, and cytomegalovirus (CMV)-sensitized sheep erythrocytes for the detection of CMV antibodies is reported. To avoid nonspecific hemagglutination, cells were fixed with glutaraldehyde or Formalin directly in whole blood. The lyophilized, CMV-sensitized erythrocytes obtained by this technique were stable up to 9 months at 37 degrees C and retained the same reactivity at fresh, CMV-sensitized cells. Indirect hemagglutination performed with lyophilized, sensitized cells was highly efficient in detecting CMV-antibodies as compared with complement fixation and enzyme immunoassay.     

Early use of indirect immunofluorescence for the detection of respiratory syncytial virus in HEp-2 cell culture

Respiratory syncytial virus is detected in cell culture by the presence of cytopathic effect. To detect RSV before cytopathic effect is usually seen, slides were evaluated retrospectively from 482 HEp-2 cell cultures on days 2-4 after inoculation. Indirect immunofluorescent staining detected RSV in 57 of 94 cultures that eventually were found positive by cytopathic effect. In an additional 19 cases that ultimately showed no cytopathic effect, RSV also was detected. In 15 of the latter cases, the presence of RSV was confirmed in the original specimen. Use of indirect immunofluorescence can be used to augment the sensitivity of cell culture for the detection of RSV because cytopathic effect may not always be evident.     

呼吸道感染患者肺炎支原体抗体检测结果分析

目的 研究肺炎支原体(MP)感染发病率与患者年龄、性别和季节的关系.方法 用被动凝集法检测呼吸道感染患者血清中肺炎支原体抗体(MP-Ab),并对2010年患者MP-Ab检测结果进行分析.结果 5年检测结果阳性率为30.10%;男、女性患者阳性率分别为30.74%、36.12%,差异有统计学意义(P<0.05);各年龄组差异有统计学意义(P<0.001),3～14岁阳性率最高;季节发病率差异无统计学意义;阳性滴度>1:640的患者占10.18%.结论 MP感染逐年增加,3～14岁儿童为高危人群,女性感染机会高于男性,全年均可发病;大多患者预后良好.     

呼吸道感染患者肺炎支原体抗体检测结果分析

目的研究肺炎支原体（MP）感染发病率与患者年龄、性别和季节的关系。方法用被动凝集法检测呼吸道感染患者血清中肺炎支原体抗体（MP-Ab）,并对2010年患者MP-Ab检测结果进行分析。结果5年检测结果阳性率为30．10％;男、女性患者阳性率分别为30．74％、36．12％,差异有统计学意义（P〈0．05）;各年龄组差异有统计学意义（P〈0．001）,3～14岁阳性率最高;季节发病率差异无统计学意义;阳性滴度〉1：640的患者占10．18％。结论MP感染逐年增加,3～14岁儿童为高危人群,女性感染机会高于男性,全年均可发病;大多患者预后良好。     

呼吸道感染患者肺炎支原体抗体检测结果分析

目的 研究肺炎支原体(MP)感染发病率与患者年龄、性别和季节的关系.方法 用被动凝集法检测呼吸道感染患者血清中肺炎支原体抗体(MP-Ab),并对2010年患者MP-Ab检测结果进行分析.结果 5年检测结果阳性率为30.10%;男、女性患者阳性率分别为30.74%、36.12%,差异有统计学意义(P<0.05);各年龄组差异有统计学意义(P<0.001),3～14岁阳性率最高;季节发病率差异无统计学意义;阳性滴度>1:640的患者占10.18%.结论 MP感染逐年增加,3～14岁儿童为高危人群,女性感染机会高于男性,全年均可发病;大多患者预后良好.     

Evaluation of an indirect fluorescent-antibody stain for detection of Pneumocystis carinii in respiratory specimens.

Two prospective studies were undertaken to evaluate a commercial indirect fluorescent-antibody (IFA) stain for the detection of Pneumocystis carinii in respiratory specimens from individuals at risk for or with the acquired immunodeficiency syndrome. The first study compared IFA with Diff-Quik (DQ; a rapid Giemsa-like stain) for detecting P. carinii in 95 induced sputa obtained from 77 asymptomatic patients who had survived one previous episode of P. carinii pneumonia and who were being treated prophylactically with aerosolized pentamidine. Only one induced sputum specimen was found to contain P. carinii; organisms were detected by both stains. The second study compared the performance of the IFA stain versus DQ, modified toluidine blue O, and Gomori methenamine silver stains for detecting P. carinii in symptomatic individuals at risk for or with acquired immunodeficiency syndrome. Of 182 specimens examined, P. carinii was detected in 105 by one or more stains; the DQ stain detected 73 (70%), the modified toluidine blue O stain detected 75 (71%), the Gomori methenamine silver stain detected 76 (72%), and the IFA stain detected 95 (90%). The IFA stain was more sensitive (P less than 0.01) than the other traditional stains for detecting P. carinii; however, a subsequent clinical evaluation revealed that a subset of IFA-positive-only specimens were from patients whose clinical symptoms resolved without specific anti-P. carinii therapy.     

Efficiency of immunofluorescence for rapid detection of common respiratory viruses.

Rapid immunofluorescence (FA) methods for the detection of common respiratory viruses were compared with culture results over a 3-year period to assess the relative efficiency of FA in a clinical laboratory setting. For respiratory syncytial virus, efficiencies were high (sensitivity, 90 to 95%; specificity, 92 to 95%). The sensitivity of FA for detection of parainfluenza virus type 1, parainfluenza virus type 3, influenza A virus, and adenoviruses ranged from 28 to 63%, but specificities for these viruses were uniformly 98 to 100%. The observations form a basis for consideration of selective reduction of routine culture procedures for specimens with initial positive rapid FA results; however, the possibility of dual viral infection in some situations must also be considered.     

Development of real-time multiplex nucleic acid sequence-based amplification for detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens

Real-time multiplex isothermal nucleic acid sequence-based amplification (NASBA) was developed to detect Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens using the NucliSens Basic Kit (bioMérieux, Boxtel, The Netherlands). Oligonucleotide primers were derived from the M. pneumoniae, C. pneumoniae, and Legionella pneumophila 16S rRNA. For real-time detection, molecular beacons were used. Specificity was established on a panel of bacterial strains. The analytical sensitivity of the assay was determined by testing dilutions of wild-type in vitro-generated RNA in water and dilutions of reference strains in lysis buffer or added to pools of respiratory specimens. Subsequently, a limited number of M. pneumoniae-, C. pneumoniae-, and L. pneumophila-positive and -negative clinical specimens were analyzed. Specific detection of the 16S rRNA of the three organisms was achieved. The analytical sensitivity of the multiplex NASBA on spiked respiratory specimens was slightly diminished compared to the results obtained with the single-target (mono) real-time assays. We conclude that the proposed real-time multiplex NASBA assay, although less sensitive than the real-time mono NASBA assay, is a promising tool for the detection of M. pneumoniae, C. pneumoniae, and Legionella spp. in respiratory specimens, regarding handling, speed, and number of samples that can be analyzed in a single run.     

Comparative studies of serological test for Mycoplasma pneumoniae on 73 cases of lower respiratory infection disease

The purpose of this report is to evaluate a test kit based on the High Density Composite Particle Agglutination Test Method (HDPA method, Newly developed by Tokuyama Soda Co., Ltd). Diagnosis of Mycoplasmosis has been done with clinical symptoms, breast x-ray examination, serum anti-M. pneumoniae antibody detection and bacteriological test result. Recently, we had the chance to use this HDPA method (IMMUNOTICLES MYCO) and compared the results with bacteriological test, complement fixation method (CF) and particle agglutination method (PA) using the cases of seventy-three (73) lower respiratory infected patients. The evaluation outcomes (positive rate, sensitivities and specificities) comparing with the conventional methods based on the clinical cultured results and clinical diagnostic results respectively are shown as follows. 1) The evaluation outcomes based on the cultured results. Forty-one (41) cases of 73 samples, we could isolate the M. pneumoniae (56.2%). a) The HDPA method is correlated with CF (r = 0.885, n = 73) and PA (r = 0.764, n = 73) respectively. b) The positive rate of HDPA, PA and CF are 45.2%, 31.5% and 20.5% respectively. 2) The evaluation outcomes based on the clinical diagnosis. a) The sensitivity of the HDPA method is 66.0% and this one is much higher than the one of CF and PA. b) The specificity of the HDPA method is 92.3%. c) The positive rate of the HDPA method is higher than the one of PA and CF even though the assay was done within seven-days. In conclusion, the HDPA method is a very sophisticated method for diagnosis of M. pneumoniae and able to be substituted to any other conventional methods.     

Production and testing of lyophilized standard preparation for diagnosis of Mycoplasma pneumoniae infection by the indirect hemagglutination test

A method for production of a reference lyophilized preparation for diagnosis of Mycoplasma pneumonia infections in the indirect hemagglutination (IHA) test has been developed. The method is based on conjugation using bi-diasotized benzidine of acrolein-treated sheep erythrocytes and ultrasonicated whole Mycoplasma antigen. After lyophilization the diagnostic preparation retained its standard properties for 1.5 years (the observation period). The sensitivity of the preparation in detection of antibody exceeded those of the CFT and the metabolism inhibition (MI) test 16--64-fold and the specificity was as good as in these tests. The diagnostic value of the preparation in the IHA was 78.1%, in the CFT--64.7%. As compared with the CFT and the MI test, the IHA with Mycoplasma diagnostic preparation is simple and reproducible.