Comparison of sputum counterimmunoelectrophoresis and culture in diagnosis of pneumococcal pneumonia.

The diagnostic value of counterimmunoelectrophoresis performed on sputum was compared with that of sputum culture. The detection of pneumococcal polysaccharide in sputum showed a better correlation with the presence of pneumococcal pneumonia than the recovery of pneumococci by culture. The authors conclude that sputum counterimmunoelectrophoresis can provide diagnostic guidance to physicians awaiting the results of sputum culture and aid in the interpretation of cultural findings.     

Comparison of three methods for detection of pneumococcal antigen in sputum of patients with community-acquired pneumonia

In a prospective study of 249 patients with community-acquired pneumonia, three tests for the detection of pneumococcal antigen in sputum were compared: a coagglutination test for detecting capsular antigens (Cap-CoA), a sandwich enzyme immunoassay (PnC-EIA) and a coagglutination test (PnC-CoA), both the latter detecting the pneumococcal C-polysaccharide common to all pneumococcal types. Sixty-three patients had culture-positive pneumococcal pneumonia, 45 pneumonia caused by other bacteria and 141 pneumonia of viral or unknown etiology. The sensitivity of Cap-CoA (63%) and PnC-CoA (65%) was somewhat higher than that of PnC-EIA (49%), but not significantly so. The specificity was 96–98% for all three methods. Using PnC-CoA 66 patients with possible pneumococcal infection were detected, the diagnosis being verified by culture in 41. Using Cap-CoA 59 such patients were detected, the diagnosis being verified in 40, and using the PnC-EIA 47 such patients were detected, the diagnosis being verified in 31. Antigen was found almost as often in non-purulent as in purulent samples, and as often in washed as in non-washed purulent samples. However, antibiotic treatment before the sputum sample was obtained resulted in significantly lower sensitivity of both PnC-CoA and Cap-CoA. This study confirms the high sensitivity and specificity of methods for pneumococcal antigen detection in sputum. Since CoA is easier and quicker to perform, and cheaper than the EIA, either PnC-CoA or Cap-CoA would seem to be the technique of choice for detection of pneumococcal antigen, whereby all sputum samples, including non-purulent samples, can be used.     

Buffy coat PCR for diagnosis of experimental pneumococcal pneumonia

An immunocompetent murine model of pneumococcal pneumonia and bacteremia was used to evaluate a PCR assay based on amplification of the pneumolysin gene. Mice were treated with trovafloxacin to determine the decline in sensitivity of PCR as lung bacterial concentrations decreased and blood cultures became sterile. Forty-three mice were studied for up to 120 h after start of antibiotic treatment. PCR of buffy coat specimens was more sensitive than PCR of plasma. Only 21% of animals had a positive blood culture, whereas 77% of PCR buffy coat assays were positive. After 48 h of therapy all blood culture specimens were sterile, whereas buffy coat PCR was positive in 57.8% of specimens. PCR of buffy coat specimens was negative in all mice colonized nasally with Streptococcus pneumoniae and in rabbits with Escherichia coli bacteremia. Our results demonstrate that our PCR technique using buffy coat specimens is highly specific for invasive pneumococcal disease and remains positive in the majority of animals for at least 48 h after start of antibiotic therapy.     

Antigen detection in oropharyngeal secretions for rapid diagnosis of pneumococcal pneumonia

To determine the value of detection of antigen in the oropharynx in the diagnosis of pneumococcal pneumonia, oropharyngeal secretions were cultured for the presence ofStreptococcus pneumoniae and tested for the presence of pneumococcal antigen. Sputum (if available) collected on the same day was also investigated for the presence of antigen. Detection of pneumococcal antigen was found to be directly related to the severity of pneumococcal carriership or infection (p<0.0001) and was not related to culture results. Patients with pneumococcal pneumonia had the highest antigen detection rate (38 %), followed by patients with pneumonia of unknown etiology (32 %) and patients with an acute lower respiratory tract infection due toStreptococcus pneumoniae (20 %). Pneumococcal carriers had a detection rate of only 9 %. Antigen could be detected in only one patient of the control groups. Although antigen detection in sputum was superior to that in oropharyngeal secretions, concordant results were obtained in 8 (40 %) and 6 (36 %) patients with pneumococcal pneumonia and pneumonia of unknown etiology respectively. The results strongly suggest that pneumococcal carriage seldom leads to a detectable level of antigen, and that antigen detection in the oropharynx appears to be of additive value in the diagnosis of pneumococcal pneumonia.     

Usefulness of pneumococcal antigen detection in pleural fluid samples by immunochromatographic assay for diagnosis of pneumococcal pneumonia

This study investigated the utility of an immunochromatographic test (ICT) for the detection of Streptococcus pneumoniae antigens in pleural fluid. Antigen was detected in 15 of 19 (79%) patients with pneumococcal pneumonia. The ICT was always negative in patients with non-pneumococcal pneumonia, but was positive in three cases with a non-infectious aetiology. In patients with pneumonia for which no pathogen was identified, antigen was detected in one of 24 pleural fluids tested. The ICT can be a valuable tool for the management of pneumonia because it can detect pneumococcal antigen in pleural effusion samples.     

Evaluation of polymerase chain reaction for diagnosis of pneumococcal pneumonia.

To test the ability of the polymerase chain reaction (PCR) to detect Streptococcus pneumoniae in blood, we generated two sets of nested primers. The first defined 559-bp and 649-bp regions of the pneumolysin gene, and the second defined 445-bp and 553-bp regions of the autolysin gene. These nucleotide segments were detected in DNAs from isolates of all 20 pneumococcal serotypes tested, but they were not detected when used to test DNAs from 41 isolates of nonpneumococcal bacteria and fungi. The sensitivity was evaluated by using purified pneumococcal DNA. We were able to detect 10 fg of S. pneumoniae DNA, or 4.3 genome equivalents. Blood samples were obtained from 16 patients with culture-proven pneumococcal bacteremia and were subjected to PCR analysis. Of eight buffy coat fractions tested, six showed reactivity in the PCR with the pneumolysin primers, and five of the eight produced the expected products when tested with the autolysin primers (sensitivities, 75 and 63%, respectively). Of the eight whole-blood specimens tested, only three produced the expected products with either set of primers. Additionally, we tested 14 samples from patients with bacteremia that were culture positive for nonpneumococcal bacterial species, and 13 were negative (specificity, 93%). This combination of sensitivity and specificity may make detection of S. pneumoniae in blood by PCR in comparison with that by blood culture a very promising alternative for a means of definitive diagnosis.     

Value of sputum culture in diagnosis of pneumococcal pneumonia.

In our laboratory, culture of sputum was extremely useful in diagnosing the etiology of pneumococcal pneumonia. Of 31 consecutive patients with bacteremic pneumococcal pneumonia, 29 (94%) had Streptococcus pneumoniae cultured from sputum. Recovery of pneumococci in culture was enhanced by anaerobic incubation as well as by a plate bile test and an optochin disk on a primary blood agar plate.     

Comparison of serological methods with PCR-based methods for the diagnosis of community-acquired pneumonia caused by atypical bacteria

Background

The diagnosis of community-acquired pneumonia (CAP) caused by Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydophila pneumoniae is traditionally based on cultures and serology, which have special requirements, are time-consuming, and offer delayed results that limit their clinical usefulness of these techniques. We sought to develop a multiplex PCR (mPCR) method to diagnosis these bacterial infections in CAP patients and to compare the diagnostic yields obtained from mPCR of nasopharyngeal aspirates (NPAs), nasopharyngeal swabs (NPSs), and induced sputum (IS) with those obtained with specifc PCR commercial kits, paired serology, and urinary antigen.

Results

A total of 225 persons were included. Of these, 10 patients showed serological evidence of L. pneumophila infection, 30 of M. pneumoniae, and 18 of C. pneumoniae; 20 individuals showed no CAP. The sensitivities were mPCR-NPS?=?23.1 %, mPCR-IS?=?57.1 %, Seeplex®-IS?=?52.4 %, and Speed-oligo®-NPA/NPS?=?11.1 %, and the specificities were mPCR-NPS?=?97.1 %, mPCR-IS?=?77.8 %, Seeplex®-IS?=?92.6 %, and Speed-oligo®-NPA/NPS?=?96.1 %. The concordance between tests was poor (kappa <0.4), except for the concordance between mPCR and the commercial kit in IS (0.67). In individuals with no evidence of CAP, positive reactions were observed in paired serology and in all PCRs.

Conclusions

All PCRs had good specificity but low sensitivity in nasopharyngeal samples. The sensitivity of mPCR and Seeplex® in IS was approximately 60 %; thus, better diagnostic techniques for these three bacteria are required.

     

Pneumolysin polymerase chain reaction for diagnosis of pneumococcal pneumonia and empyema in children

Streptococcus pneumoniae is the most important cause of childhood pneumonia and empyema, yet the diagnosis of pneumococcal infections by conventional methods is challenging. In this study, the clinical value of the pneumolysin-targeted real-time polymerase chain reaction (PCR) method for the diagnosis of pneumococcal pneumonia and empyema was evaluated with 33 whole blood samples and 12 pleural fluid samples. The analytical sensitivity of the PCR assay was 4 fg of pneumococcal DNA, corresponding to two genome equivalents of pneumococcal DNA per reaction. The PCR assay correctly detected all clinical isolates of S. pneumoniae tested, whereas all nonpneumococcal bacterial organisms tested were negative by PCR. In a clinical trial, S. pneumoniae was detected by PCR in the pleural fluid of 75% of children with empyema, increasing the detection rate of pneumococcus almost tenfold that of pleural fluid culture. However, in whole blood samples, PCR detected S. pneumoniae in only one child with pneumonia and one child with pneumococcal empyema and failed to detect S. pneumoniae in three children with blood cultures positive for S. pneumoniae. The present data indicate that pneumolysin-targeted real-time PCR of pleural fluid is a valuable method for the etiologic diagnosis of pneumococcal empyema in children. The ease and rapidity of the LightCycler technology (Roche Diagnostics, Mannheim, Germany) make real-time PCR an applicable tool for routine diagnostics. In the evaluation of blood samples, blood culture remains the superior method for the diagnosis of bacteremic pneumococcal disease.     

A critical study of immunological methods for pregnancy diagnosis

Immunological methods of pregnancy diagnosis using both tanned red cell and latex particles are compared in a series of women (up to the fourteenth week of gestation) and also in negative controls. The tanned red cell test showed slightly more accurate and sensitive results throughout the trial. In 31 samples also submitted for the Hogben test the immunological test showed marginal superiority. It is concluded that immunological methods are reliable for the diagnosis of pregnancy at least between the fifth and the fourteenth weeks of gestation.     

Validation of immune-complex enzyme immunoassays for diagnosis of pneumococcal pneumonia among adults in Kenya

The efficacy of pneumococcal vaccines in protecting against pneumococcal pneumonia can feasibly be measured only with a diagnostic technique that has a high specificity (0.98 to 1.00) and a sensitivity greatly exceeding that of blood cultures (>0.2 to 0.3). In this context immune-complex enzyme immunoassays (EIAs) offer a novel, convenient diagnostic method, and we have investigated three such assays with appropriate study populations in Kenya. Sera from 129 Kenyan adults with pneumococcal pneumonia and 97 ill controls from the same clinics, but without pneumococcal disease syndromes, were assayed with immune-complex EIAs for pneumolysin, C-polysaccharide, and mixed capsular polysaccharides (Pneumovax II). At an optical density (OD) threshold yielding a specificity of 0.95, the sensitivities (95% confidence intervals) of the assays were 0.22 (0.15 to 0.30), 0.26 (0.19 to 0.34), and 0.22 (0.15 to 0.29), respectively. For pneumolysin immune complexes, human immunodeficiency virus (HIV)-positive patients had a higher mean OD than HIV-negative patients (639 versus 321; P < 0.0001), but stratification by HIV infection status did not alter the performance of this test. Combining the results of all three EIAs did not enhance the diagnostic performances of the individual assays. In Kenyan adults the sensitivities of the immune-complex EIAs could exceed that of blood cultures only at levels of specificity that were insufficient for the performance of vaccine efficacy studies.     

Comparison of enzyme-linked immunosorbent assay with coagglutination and latex agglutination for rapid diagnosis of pneumococcal pneumonia by detecting antigen in sputa

A sandwich enzyme-linked immunosorbent assay detecting the species-specific pneumococcal C polysaccharide was compared to latex agglutination and a coagglutination test which detected capsular pnenmococcal antigens in sputum specimens with regard to specificity and sensitivity. Specimens from 52 patients with clinical and radiological evidence for pneumonia were tested. Twenty-one patients with Streptococcus pneumoniae isolated in sputum and 31 patients with a non-pneumococcal etiology were included. The predictive values for a positive test by enzyme-linked immunosorbent assay was 0.91 and for a negative test 0.97, by latex agglutination 0.90 and 0.91, and by coagglutination 0.84 and 0.85 respectively; these values did not show a statistically significant difference. Whereas agglutination tests are technically more simple and can be performed more rapidly, the enzymelinked immunosorbent assay has the advantage of detecting pneumococcal C polysaccharide, an antigen common to all pneumococci. Thus it provides an interesting alternative to tests based on serum containing antibodies to all 83 different capsular polysaccharides.     

Comparison of microbiologic assay methods for hemodialysis fluids.

To help prevent pyrogenic reactions and bacteremia in hemodialysis patients, the Association for the Advancement of Medical Instrumentation and the Centers for Disease Control recommend microbiologic assay of hemodialysis fluids at least monthly. Five commercially available assay systems were evaluated by using the membrane filtration technique with standard methods agar and trypticase soy agar as the standards for comparison. Each assay system was challenged with dialysate and reverse-osmosis water from local dialysis centers, aqueous suspensions of eight laboratory strains of gram-negative bacilli and nontuberculous mycobacteria, and a mixed microbial flora inoculated into reverse-osmosis water and laboratory-prepared dialysate. Mean viable counts from triplicate samples were obtained after incubation at 37 degrees C for up to 72 h. The efficiency of recovery varied with the specific type of microbial challenge. The SPC water sampler (Millipore Corp., Bedford, Mass.) was the most consistent in obtaining the highest viable counts. Other commercial systems were comparable to each other in overall performance. All assay systems tested provided an acceptable balance between microbial recovery and required sampling time, equipment, and expertise.     

The usefulness of adenosine deaminase determination in biological fluids for tuberculosis diagnosis

Tuberculosis may affect several organs and its prevalence is continuously increasing. Laboratory diagnosis still remains difficult. Adenosine deaminase (Ada) is an enzyme which contributes to purine metabolism and its presence in lymphocyte, monocyte and macrophage cells is associated with T cells mediated immunity. Many studies have shown the usefulness of Ada determination in various biological fluids for the diagnosis of tuberculosis. In pleural fluid, cutoff vary from 33 to 48 U/L, with sensitivity higher than 80% and specificity near 100%. In peritoneal fluid the cutoff value is 30 U/L. In cerebrospinal fluid, the value of 7 U/L can make discriminate negative and positive cases with a good sensitivity and specificity. The data from the literature show that 50 U/L in pericardic fluid is a reliable threshold for tuberculosis diagnosis. Ada determination in serum is not as relevant as in others fluids because of its low specificity. Ada measurement in biological fluids, easy and not expensive, may be add to other biological tests for tuberculosis diagnosis.     

Comparison of three staining methods for detecting microsporidia in fluids.

Calcofluor white 2MR, modified trichrome blue, and indirect immunofluorescent antibody (IFA) staining methods were evaluated and compared for detecting microsporidia in stool. Serial 10-fold dilutions of Encephalitozoon (Septata) intestinalis were prepared in three formalinized stool specimens or in Tris-buffered saline. Ten-microliter aliquots were smeared onto glass slides, fixed with methanol, stained, and read by at least three individuals. The results indicated that the calcofluor stain was the most sensitive method, required approximately 15 min to perform, but did generate some false-positive results due to similarly staining small yeast cells. The modified trichrome blue stain was nearly as sensitive as the calcofluor stain and allowed for easier distinction between microsporidia and yeast cells. This stain, however, required approximately 60 min to perform. The IFA stain with polyclonal murine antiserum against E. intestinalis was the least sensitive of the methods and required approximately 130 min to perform. The lower limit of detection with the calcofluor and modified trichrome stains was a concentration of about 500 organisms in 10 microliters of stool to detect one microsporidian after viewing 50 fields at a final magnification of x1,000. Reliability was also addressed by use of 74 stool, urine, and intestinal fluid specimens, 50 of which were confirmed for the presence of microsporidia by transmission electron microscopy (TEM). All TEM-positive specimens were detected by calcofluor and modified trichrome blue staining. Ten specimens were not detected by the IFA stain. An additional seven TEM-negative specimens were read positive for microsporidia with the calcofluor stain, and of these, five also were read positive with the modified trichrome blue stain. The resulting diagnostic paradigm was to screen specimens with the calcofluor stain and to confirm the results with the modified trichrome stain. IFA, which was less sensitive, may become useful for microsporidian species identification as specific antibodies become available.     

Usefulness of urinary antigen detection by an immunochromatographic test for diagnosis of pneumococcal pneumonia in children

We evaluated an immunochromatographic assay detecting pneumococcal antigen in urine samples from children diagnosed with pneumococcal pneumonia. The sensitivity and specificity of the immunochromatographic test with nonconcentrated urine (NCU) were 86.7 and 62.9%, respectively; with concentrated urine (CU), they were 100 and 11.7%, respectively. Pneumococcal antigen was also detected in 42.5% of NCU and 87.1% of CU samples from nasopharyngeal carriers. This is a nonspecific test for the diagnosis of pneumococcal pneumonia in children, particularly the very young.     

A comparison between two methods for measuring tumor necrosis factor in biological fluids

The current study was undertaken to compare two methods for the efficiency of measuring tumor necrosis factor (TNF-α) in biological fluids, which is species undependent, reliable, sensitive, simple and not expensive. We have compared the MTT tetrazolium cytotoxic assay [1,2] and the³H-thymidine (³H-TdR) incorporation cytostatic assay for measuring the anti-tumor activity of human recombinant TNF-α, of human colonic tissue and of supernatants ofin vitro stimulated human and rat peritoneal macrophages. Two target cell-lines, namely murine myelomonocytic leukaemia WEHI-164- and L-929-transformed murine fibroblast cell-lines, were used in the MTT assay. The L-929 line was also used in the³H-TdR assay. WEHI-164 was more sensitive than the L-929 cell-line in the MTT cytotoxic assay. Furthermore, the MTT assay was more sensitive to TNF-α than the³H-TdR assay. Both methods can be used for the detection of anti-tumor activity in biological fluids but the MTT cytotoxic method has the advantage of being more sensitive and more simple.