Diagnostic value of different laboratory methods in the diagnosis of pneumonia caused by Haemophilus influenzae type B

Comparative analysis of the diagnostic value of different laboratory methods in the diagnosis of H. influenzae b (Hib) pneumonia in children (bacteriological method, latex agglutination, counter immunoelectrophoresis, the passive hemagglutination test and the enzyme immunoassay (EIA) was carried out. EIA proved to be the most informative method for the diagnosing Hib pneumonia. EIA makes it possible to detect specific Hib antigens in different clinical materials in 48.8% of cases, as well as high titers of antibodies to mis infective agent in 61.7% of cases. The authors propose the unified criteria of the laboratory diagnosis of Hib infection in children.     

Simple and rapid F+ coliphage culture, latex agglutination, and typing assay to detect and source track fecal contamination

Simple, rapid, and reliable fecal indicator tests are needed to better monitor and manage ambient waters and treated waters and wastes. Antibody-coated polymeric bead agglutination assays can fulfill these needs and are inexpensive and portable for nonlaboratory settings, and their reagents can be stored at ambient temperatures for months. The goal of this study was to develop, optimize, and validate a rapid microbial water quality monitoring assay using F+ coliphage culture, latex agglutination, and typing (CLAT) to detect F+ coliphage groups with antibody-coated particles. Rapid (180 min) F+ coliphage culture gave comparable results to those with the 16- to 24-h culture time used in EPA method 1601 and was amenable to CLAT assay detection. CLAT was performed on a cardboard card by mixing a drop of coliphage enrichment culture with a drop of antibody-coated polymeric beads as the detection reagent. Visual agglutination or clumping of positive samples occurred in <60 seconds. The CLAT assay had sensitivities of 96.4% (185/192 samples) and 98.2% (161/164 samples) and specificities of 100% (34/34 samples) and 97.7% (129/132 samples) for F+ RNA and DNA coliphages, respectively. CLAT successfully classified F+ RNA coliphages into serogroups typically obtained from human (groups II and III) and animal (groups I and IV) fecal sources, in similar proportions to those obtained with a nucleic acid hybridization assay. This novel group-specific antibody-based particle agglutination technique for rapid and simple detection and grouping of F+ coliphages provides a new and improved tool for monitoring the microbiological quality of drinking, recreational, shellfishing, and other waters.     

Comparative evaluation of whole blood D-dimer test to plasma D-dimer test for diagnosis of disseminated intravascular coagulation

Three rapid D-dimer test methods were compared for the diagnosis of acute disseminated intravascular coagulation (DIC). These were (a) SimpliRED, an autologous red cell agglutination assay. (b) DIMERTEST latex agglutination assay, containing monoclonal antibody DD-3B6/22(6), and (c) D-DI latex agglutination assay containing mouse anti-human D-dimer monoclonal antibodies. The D-DI latex method having higher sensitivity (100%) and specificity (81%) in clinically acute DIC was postulated as the gold standard and compared with the other two methods. The results suggest that D-DI latex agglutination assay containing mouse anti-human D-Dimer monoclonal antibodies are the better assay methods amongst all the three kits analyzed. It is advisable to look for the nature of the antibody used to coat the latex particles in plasma based kits. In emergency setting RBC kits may be of some use as rapid diagnosis is advantageous.     

New techniques in rapid viral diagnosis

Abstract The development of new diagnostic techniques in immunology and molecular biology during the last two decades has opened up new possibilities for rapid viral diagnosis. Solid phase immunoassays for antigen and antibody detection are now widely used in diagnostic settings. Several novel techniques have been introduced and have led to commercially available tests. Diagnostic methods using nucleic acid amplification procedures are already applied in research laboratories and will be commercialized soon. Biosensor-based diagnostic techniques have the potential of generating a result nearly instantaneously and it has become possible to monitor kinetic processes. Automatization and simplified procedures are needed to allow diagnostic tests to be performed soon after the sample has been obtained from the patient. In order to evaluate the new procedures and avoid false results, rigorous quality control in diagnostic virology will have to be instituted.     

Comparative evaluation of two commercially available antigen enzyme immunoassays (EIA) for the detection of Legionella pneumophila urinary antigen in frozen non-concentrated urine samples

We evaluated two commercial enzyme immunoassay kits, Binax EIA (for detection of soluble antigen of Legionella pneumophila serogroup 1) and Biotest EIA (for detection of antigens of Legionella pneumophila serogroups and other Legionella spp.) in order to introduce this test routinely for the diagnosis of Legionnaires' disease (LD) in our Laboratory. Frozen non-concentrated urine samples belonging to 45 patients with and without LD were tested. The sensitivity of Binax EIA and Biotest EIA was 47.4% and 42.1% respectively, the specificity was 95% by both tests. Biotest did not detect antigen from a patient with culture-proven infection of L. pneumophila serogroup 6. The detection of urinary antigen by both EIA tests is a useful tool for rapid diagnosis of LD, especially when samples are unavailable for culture; the sensitivity may be increased if the assay is performed on unfrozen and concentrated samples.     

Specific latex preparations in the etiological diagnosis of suppurative bacterial meningitis

The results of the evaluation of the diagnostic latex preparations Bactigen, manufactured by Wampole Laboratories (USA) and intended for the detection of meningococcal antigens, serogropus A, B, C, Y, pneumococcal polyantigens and type b Haemophilus influenzae antigens in the spinal fluid and blood of patients with meningococcal infection and purulent bacterial meningitides, are presented. The pathological material was studied by traditional methods and by the latex agglutination (LAG) test. 522 LAG tests were made, including 414 tests for meningococcal infection, 60 tests for pneumococcal infection and 48 tests for type b H. influenzae. The results of this study revealed that the latex preparations were highly specific with respect to type b H. influenzae antigens and meningococcal antigens (false positive reactions constituted 0.96%). The simplicity of the test and the rapid techniques making it possible to obtain results within 30-40 minutes indicate good prospects of using the LAG test in laboratory practice.     

RS virus diagnosis: comparison of isolation, immunofluorescence and enzyme immunoassay

Two techniques for rapid diagnosis, immunofluorescence (IFAT) and enzyme immunoassay (EIA), have been compared with virus isolation in tissue culture for the detection of respiratory syncytial virus (RSV) in specimens of nasopharyngeal secretions. The specimens were obtained from children under five years of age suffering from acute respiratory illness, during a period of six months from January to June 1982. Of 471 specimens examined 54 (11.5%) were positive by virus isolation and 180 (38.2%) were positive by immunofluorescence. The bacterial contamination of inoculated tissue cultures unfortunately prevented the isolation of virus from many samples. Specimens from 216 children were tested to compare enzyme immunoassay and immunofluorescence. Of these 60 (27%) were positive by EIA and 121 (56%) were positive by IFAT. Our results suggest that the EIA technique although highly specific is rather insensitive. This may be because by the time these tests were done the original nasopharyngeal secretions were considerably diluted and contained more mucus fragments than the cell suspension used for IFAT. Of the three techniques, IFAT gives the best results although EIA may be useful where IFAT is not possible.     

The results of using the molecular hybridization of nucleic acids in the complex study of nasopharyngeal smears from patients with influenza and other acute respiratory diseases

A complex study of samples obtained from patients with influenza and other acute respiratory diseases has revealed that the laboratory methods used in this study can be rated in the following order according to their sensitivity: isolation of the virus in chick embryos, analysis of seroconversions in the hemagglutination inhibition test, immunofluorescent determination of viral antigens, determination of viral antigens by enzyme immunoassay (EIA), detection of RNA-containing viral structures by means of molecular hybridization. From the point of view of the possibility of documenting influenza A in patients, the best results are achieved by the combination of molecular hybridization and EIA techniques: 90% and more of all cases. A rational scheme for the examination of samples obtained from patients with a view to epidemiological study, including both traditional and new rapid diagnostic methods, is proposed.     

A simple nucleic acid hybridization/latex agglutination assay for the rapid detection of polymerase chain reaction amplicons

We have developed a new method for the detection of nucleic acid hybridization, based on a simple latex agglutination test that can be evaluated by the unaided eye. Nucleic acid, e.g., a polymerase chain reaction (PCR) product, is denatured and incubated with polystyrene beads carrying covalently bound complementary oligonucleotide sequences. Hybridization of the nucleic acids leads to aggregation of the latex particles, thereby verifying the presence of target sequence. The test is performed at room temperature, and results are available within 10 min. As a proof of principle, the hybridization/latex agglutination assay was applied to the detection of purified PCR fragments either specific for Salmonella spp. or a synthetic sequence, and to the detection of Salmonella enterica in artificially contaminated chicken samples. A few nanograms of purified PCR fragments were detectable. In artificially contaminated chicken samples, 3 colony-forming units (cfu)/25 g were detected in one of three replicates, and 30 cfu/25 g were detected in both of two replicates when samples for PCR were taken directly from primary enrichment, demonstrating the practical applicability of this test system. Even multiplex detection might be achievable. This novel kind of assay could be useful for a range of applications where hybridization of nucleic acids, e.g., PCR fragments, is to be detected.     

Evaluation of a latex agglutination test for rapid detection of rotavirus in faecal specimens

Two methods for detecting rotaviruses (latex agglutination, electron microscopy) have been compared on 80 faecal samples. These samples were obtained from infants between the age of four and 30 months hospitalized for acute gastroenteritis in the Children's Hospital, Karl Marx University at Leipzig, in 1982. Complete agreement among the two techniques was found in 75 specimens. Sensitivity of latex agglutination could be estimated at 95%, the specificity also at 95%. Only one sample reacted nonspecifically. Performance of the latex agglutination proved quite simple. The results indicate that latex agglutination is suitable for rapid screening of rotavirus induced gastroenteritis in clinical practice thus enabling the rate of nosocomial rotavirus infections in children's hospitals to be reduced.     

Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors

In situ hybridization (ISH) is a powerful technique for localizing specific nucleic acid sequences (DNA, RNA) in microscopic preparations of tissues, cells, chromosomes, and linear DNA fibers. To date, a wide variety of research and diagnostic applications of ISH have been described, making the technique an integral part of studies concerning gene mapping, gene expression, RNA processing and transport, the three-dimensional organization of the nucleus, tumor genetics, microbial infections, and prenatal diagnosis. In this review, I first describe the ISH procedure in short and then focus on the currently available non-radioactive probe-labeling and cytochemical detection methodologies that are utilized to visualize one or multiple different nucleic acid targets in situ with different colors. Special emphasis is placed on the procedures applying fluorescence and brightfield microscopy, the simultaneous detection of nucleic acids and proteins by combined ISH and immunocytochemistry, and, in addition, on the recent progress that has been made with the introduction of signal amplification procedures to increase the detection sensitivity of ISH. Finally, a comparison of fluorescence, enzyme cytochemical, and colloidal gold silver probe detection systems will be presented, and possible future directions of in situ nucleic acid detection will be discussed. Accepted: 9 June 1999     

Rapid latex agglutination test for detection of staphylococcal enterotoxins A to E that uses high-density latex particles.

A rapid reversed passive latex agglutination method that uses high-density latex particles for the detection of staphylococcal enterotoxins (SE) A to E was developed. It took 3 h for incubation, much less than the 16 h needed with a customary latex agglutination test for SE detection such as a commercial test kit (SET-RPLA; Denka Seiken Co. Ltd., Tokyo, Japan). The rapid test was shown to be highly specific and sensitive for SE detection (detection limit, about 0.5 ng of SE per ml), comparable to the SET-RPLA test. The rapid test was also efficient in SE detection in foods and culture supernatants of staphylococcal strains, similar to the SET-RPLA test. This showed that a rapid test with high-density latex particles is fully reliable for use.     

Rapid latex agglutination test for detection of staphylococcal enterotoxins A to E that uses high-density latex particles

A rapid reversed passive latex agglutination method that uses high-density latex particles for the detection of staphylococcal enterotoxins (SE) A to E was developed. It took 3 h for incubation, much less than the 16 h needed with a customary latex agglutination test for SE detection such as a commercial test kit (SET-RPLA; Denka Seiken Co. Ltd., Tokyo, Japan). The rapid test was shown to be highly specific and sensitive for SE detection (detection limit, about 0.5 ng of SE per ml), comparable to the SET-RPLA test. The rapid test was also efficient in SE detection in foods and culture supernatants of staphylococcal strains, similar to the SET-RPLA test. This showed that a rapid test with high-density latex particles is fully reliable for use.     

Diagnóstico microbiológico de las micosis invasoras

The prognosis of invasive fungal infections (IFI) depends on the speed of diagnosis and treatment. Conventional diagnostic methods are of low sensitivity, laborious and too slow, leading to the need for new, faster, and more efficient diagnostic strategies.There are several techniques for diagnosing a candidemia that are faster than the conventional blood culture (BC). Once yeast growth in BC is detected, species identification can be speeded up by mass spectrometry (30 minutes), commercialised molecular techniques (60-80 minutes) or fluorescent in situ hybridization (90 minutes). The combined detection of biomarkers (antimicellium, mannan and anti-mannan or β-glucan) has shown to be of greater use than their individual use. Commercialised nucleic acid amplification techniques (Septifast®, T2Candida®) are very reliable alternatives to BC. The detection of the capsular antigen of Cryptococcus, by means of latex agglutination or immuno-chromatography, is a valuable technique for cryptococcosis diagnosis.Direct microscopic examination and culture of representative specimens is used for the conventional diagnosis of IFI by filamentous fungi. Detection of galactomannan and β-glucan are considered diagnostic criteria for probable invasive aspergillosis and probable IFI, respectively, despite the lack of specificity of the latter. The detection of fungal volatile organic compounds in breath is an interesting diagnostic strategy in pulmonary infections. Although widely used, nucleic acid detection techniques are not considered diagnostic criteria for IFIs caused by moulds in consensus documents, due to their lack of standardisation. However, they are the only alternative to culture methods in invasive infections by Scedosporium/Lomentospora, Fusarium, zygomycetes, or dematiaceous fungi.     

Comparative Evaluation of Five Serological Methods for the Diagnosis of Sporotrichosis

The diagnosis of sporotrichosis can be time consuming. Serological procedures could facilitate the rapid and accurate diagnosis of this disease. A slide latex agglutination (SLA) test for sporotrichosis was developed and compared with the tube agglutination (TA), complement fixation (CF), and immunodiffusion (ID) tests in the serological study of 80 proven human cases of sporotrichosis representing the cutaneous, subcutaneous, and extracutaneous forms of the disease. In addition, the indirect fluorescent antibody (IFA) technique was applied to 61 case sera. In the SLA test, latex particles sensitized with culture filtrate antigens from the yeast form of Sporothrix schenckii (B 959) detected 94% of the cases, as compared to 96% of the cases detected by the TA test, 68% by the CF test, and 56% by the ID test. The IFA test detected 90% of the 61 cases. The SLA and ID tests were specific, showing no reactions with sera from 86 persons with no disease or with diseases other than sporotrichosis. Because of its sensitivity, specificity, ease of performance, and ability to provide results in 5 min, the SLA test is highly recommended for routine use in the clinical laboratory.     

Immunoenzyme method in the diagnosis of human brucellosis

The optimum conditions for the determination of specific antibodies in the sera of brucellosis patients by means of enzyme immunoassay (EIA) have been selected. The comparative study of the specificity and sensitivity of EIA and other serological tests has demonstrated that EIA has high diagnostic effectiveness in the diagnosis of acute and chronic brucellosis. The presence of direct correlation between the results of EIA and Coombs' test is observed, which is indicative of the capacity of EIA for detecting both complete and incomplete specific antibodies. It should be pointed out that in all cases the titer of specific antibodies in EIA has been found to be 5-16 times higher than in Coombs' test, the passive hemagglutination test, and agglutination test.     

Use of microcapsular leptospiral antigens for detecting specific antibodies

The sensitivity of microcapsular leptospiral antigens, produced by Japan Lyophilization Laboratory and intended for use in tests for the detection of antibodies to leptospires in the sera of experimentally immunized laboratory animals, were studied. The comparative study of the microcapsular agglutination (MCA) test and other serological tests, such as the microagglutination (MA) test and the indirect enzyme immunoassay (EIA), was made. The leptospiral antigens under study were found to actively react with serospecific and group-specific antibodies. In infected guinea pigs and rabbits specific antibodies could be detected from days 3-4 in the MCA test and only from days 5-7 in the MA test. The average antibody level determined by titration in the MCA test was 3.3 times higher and in indirect EIA, 4.3 times higher than that determined by titration in the MA test. These data make it possible to recommend the use of microcapsular leptospiral antigens for the early diagnosis of leptospirosis.     

Comparative evaluation of the sensitivity of immunofluorescence methods, immunoenzyme analysis and the lectin test for the rapid diagnosis of influenza

Comparative study of the sensitivities of immunofluorescent microscopy (IFM), enzyme immunoassay, (EIA), and lectin test (LT) in the detection of influenza virus antigen in nasopharyngeal washings from patients with influenza, acute respiratory diseases, pneumonia, and laryngitis has been carried out. EIA modification (used in this study) based on the detection of a complex of viral core proteins (M + RNP) has been shown to be no less sensitive than IFM and suitable for use in the rapid diagnosis of influenza. It can be used in combination with other methods. The optimum time for collecting the washings off is day 2 from the disease onset for analysis by EIA technique and day 4 for LT.     

Screening for plant lectins by latex agglutination tests

The latex agglutination test has been applied as a detection system for lectins, the method being especially useful in locations where the dependence on blood for hemagglutination tests could be minimised. The binding of various glycoproteins and sugars individually to the latex particles facilitated the agglutination with lectins having varying sugar specificities. The glycoproteins used were ovalbumin, horseradish peroxidase, porcine mucin and fetuin, while N-acetylglucosamine, N-acetylgalactosamine comprised the sugars used for binding to latex. The sensitivity of the latex agglutination tests was comparable with that of hemagglutination tests. Sugar binding specificity of the lectins could also be determined by inhibition of the agglutination in the presence of corresponding free sugars. The method proved to be useful in screening crude seed extracts for the presence of lectins.     

Effect of formulation on the viability of Metarhizium anisopliae conidia

A slide agglutination test using antibody-sensitized latex particles was developed for the specific detection in the early infection of the flacherie virus of the silkworm, Bombyx mori. With this test, 0.63 μg/ml of virus protein could be detected. The tests was completed within 5 min. Extracts from flacherie virus-infected silkworm larvae agglutinated latex particles specifically, while there was no agglutination by extracts of normal and nuclear polyhedrosis virus-infected silkworm larvae. The results showed that the sensitivity and simplicity of this technique for the detection of flacherie virus were greater than those of conventional serological techniques such as the immunofluorescence test and the immunodiffusion test.