Limitations of the direct immunofluorescence test for antibody-coated bacteria in determining the site of urinary tract infections in children.

The results of the direct immunofluorescence test for antibody-coated bacteria to determine the site of a urinary tract infection do not always correlate with the clinical data. When this test was performed on urine specimens from 282 children with significant bacteriuria, false-negative and false-positive results were observed in 20% (19/94) and 52% (19/188) of the specimens. Contamination of the specimen during collection and the presence of Fc receptors (receptors for the crystallizable fragment of the immunoglobulin molecule) on the surface of some strains of Staphylococcus aureus yielded false-positive results, and stools and vaginal secretions were shown to be potential sources of antibody-coated bacteria. It is suggested that for children this test be run on urine collected by bladder puncture. The use of conjugated anti-IgG antiserum containing only F(ab'')2 (the antigen-binding fragments of the IgG molecule) is also recommended to eliminate false-positive results due to the presence of Fc receptors on the bacterial surface.     

The rapid demonstration and presumptive identification of Listeria monocytogenes in food using monoclonal antibodies in a direct immunofluorescence test (DIFT)

Two monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC) were successfully used in a direct immunofluorescence test (DIFT) to demonstrate listeria in seven samples of soft cheese where Listeria monocytogenes had been cultured by conventional techniques. Using DIFT, listeria was not detected in 20 cheese samples from which L. monocytogenes had not been isolated, or was present in low numbers (< 10²/g). The DIFT was also used to presumptively identify > 90% of strains of L. monocytogenes isolated from food and cultured on Modified McBride agar or Blood agar. Less than 10% of strains of other species of listeria would be misidentified when grown on these media. All tests were carried out within 2 h.     

Technical note on the demonstration of rheumatoid factor by immunofluorescence

The authors present a new technic to point out the rheumatoid factor by immunofluorescence and compare this technic and the others to classical technics of agglutination. This technic allows to avoid the absorption of rheumatoid factor in the serological reactions using the immunofluorescence.     

Preparation of monoclonal antibodies cross-reactive with orthopoxviruses and their application for direct immunofluorescence test

Variola virus (smallpox virus), vaccinia virus (VV), cowpox virus (CPV) and ectromelia virus (EV) belong to the genus Orthopoxvirus of the family Poxviridae. To establish the possible diagnosis for smallpox infection, monoclonal antibodies (MAbs) against VV and CPV were produced. The cross-reactivity of seven MAbs with cells infected with various strains of the orthopoxviruses (CPV, VV and EV) was confirmed by an immunofluorescence (IF) test and other immunological analyses. Four and three MAbs reacted with the common antigen of all poxviruses (probably NP antigen) and the antigen involved in neutralization, respectively. We developed the IF test using these MAbs. The direct IF test required only 45 min to perform. Smallpox infection is now eradicated, but it is important to prepare for the diagnosis of smallpox in an emergency. The direct IF assay using MAbs cross-reactive with orthopoxviruses is rapid, simple, specific, applicable for multiple samples, and will make it possible to screen for and detect orthopoxviruses that include variola virus with tissue impression smears from skin lesions in most laboratories or institutes.     

Simultaneous demonstration of multiple antigens by indirect immunofluorescence or immunogold staining

Summary Available techniques for light and electron microscopical double immunocytochemical staining are all associated with certain problems. We have developed a novel multiple staining procedure, which allows use of antibodies of differing specificities, raised in the same species (e.g. rabbit). Its essential features include 1) saturation of antigenic epitopes on the first layer primary antiserum by second (fluorophor- or gold-) labelled anti-IgG antibodies and 2) denaturation of free anti-IgG binding sites by formaldehyde vapour treatment. Various combinations of gastrin, somatostatin, glucagon, ACTH, growth hormone and enkephalin/endorphin antibodies have been tested at the light and electron microscopical level and have been found to give highly reproducible double- and triple-staining results. The technique has also been evaluated by use of cytochemical paper models. The method is simple and very useful for multiple staining of a wide variety of antigens.     

A glutinous rice culture medium for demonstration of chlamydospores of candida albicans

Thirty-four recent isolates ofCandida albicans from clinical material were cultured on glutinous rice agar at 21 pH values ranging from 2.2 to 11.9. After incubation at 25°C all isolates produced chlamydospores on this medium at pH values from 6.6 to 8.0 with an optimum pH of 7.1. Nineteen stock cultures and all recent isolates ofCandida albicans were used to compare the new glutinous rice agar with 9 other culture media recommended for chlamydospore formation. The results indicated that the new medium was superior in terms of (1) economy, (2) rapid production of chlamydospores, (3) transparency and (4) ease of investigation by direct microscopic examination.

---

Zusammenfassung Vierunddreißig jüngst isolierte Stämme vonCandida albicans aus klinischem Material sind auf Glutin-Reisagar innerhalb 21 pH-Werte vom 2.2 bis 11.9 gezüchtet worden. Nach Inkubation bei 25°C haben alle Stämme auf diesem Medium bei den Werten von pH 6.6 bis 8.0 Chlamydosporen produziert mit dem Optimum bei pH 7.1. Neunzehn Stammkulturen und alle jüngst isolierten Stämme vonC. llbicans sind verwendet worden um den neuen Glutin-Reisnährboden mit neun anderen, empfohlenen Nährböden fur Chlamydosporen-Produktion zu vergleichen. Die Ergebnisse zeigten, daß der neue Nährboden in folgenden Beziehungen vortrefflicher war: 1) Wirtschaftlichkeit; 2) rasche Chlamydosporen-Produktion; 3) Durchsichtigkeit; 4) Leichtigkeit bei direkter mikroskopischer Untersuchung.

     

The use of trypan blue for counterstaining in the Sepharose bead immunofluorescence test. The application of the test for the demonstration of primate retrovirus-specific antibodies and antigens.

The Sepharose bead immunoflurorescence test was performed by counterstaining the beads with trypan blue. This results in a red staining of negative beads which allows an easy distinction from positive green-fluorescent beads. Sepharose beads conjugated with viral proteins or antiviral antibodies were used to demonstrate Mason-Pfizer monkey virus (MPMW)- and simian sarcoma virus (SSV) - specific antigens or antibodies. The test shows a high sensitivity and specificity and needs a small amount of material.     

Development and evaluation of a rapid identification test for candida albicans

Summary A new technique for the rapid identification ofC. albicans has been developed and evaluated. This yeast can be identified in one hour by the formation of germ tubes after inoculation in 1/2 ml of human or animal plasma, and commercial plasma substitutes.C. albicans also forms germ tubes within 2 to 4 hours after inoculation in human serum and incubation at 37° C.Filamentation ofC. albicans in these blood derivatives is a reliable method for the identification of this yeast. It is more rapid than the assimilation and fermentation sugar tests and chlamydospore formation.Assimilation and fermentation sugar tests are used to identify those isolates ofCandida that fail to produce filaments in plasma or serum.     

Antibody detection and cross-reactivity among species and serogroups of Legionella by indirect immunofluorescence test

The purpose of the present study was to determine possible cross-reactivity between different serogroups or between different species by means of bacterial smears of individual L. pneumophila serogroups and several non-pneumophila Legionella spp. Using BIOCHIP slide we found a considerable frequency of cross-reactions with L. pneumophila serogroup 12 (88.4%) therefore BIOCHIP slide proved as useful method for identification of cross-reactivity between members of the Legionnellaceae.     

The demonstration,by immunofluorescence,of Legionella pneumophila organisms in human lung tissue embedded in epoxy resin

Summary Legionella pneumophila serogroup 1 has been demonstrated by indirect immunofluorescence in human lung tissue fixed in 10% formal saline and embedded in epoxy resin. The organism was visualised using Rabbit serogroup 1 antibody and Sheep anti rabbit fluorescein conjugate. The fluorescent labelled organism was found distributed throughout the tissue with focal areas in alveolar spaces and also visualised by electron microscopy in the same tissue. This method therefore enables specific identification of the L. pneumophila organism with the antiserum and also affords the opportunity of studying the bacterium ultrastructurally in the same tissue.     

The application of direct immunofluorescence to intraoperative neurosurgical diagnosis

A diagnostic problem can occur at the time of intraoperative consultation of neurosurgical tumors as to whether the tumor is of neuroectodermal origin or whether it represents an epithelial metastasis from another site. Intraoperative diagnoses based on hematoxylin and eosin stained frozen sections are often later confirmed by immunocytochemical analysis of formalin-fixed, paraffin-embedded tissue sections that are not available at the time of surgery. The objective of the current study was to demonstrate that the application of direct immunofluorescence to the intraoperative diagnosis of neurosurgical tumors would provide unequivocal, and nearly immediate results. This report describes a new application of an existing technique for an optimized, rapid procedure utilizing direct immunocytochemistry with fluorescence-labeled primary antibodies to analyze surgical biopsies intraoperatively. The examination of five neurosurgical biopsies established a neuroectodermal origin of three tumors via immunolabeling for glial fibrillary acidic protein (GFAP) and lack of labeling with keratin markers, whereas several metastatic lung carcinomas were identified by immunostaining for keratin, but not GFAP, markers. The results of the direct immunolabeling method were unequivocal and required only minutes. The same diagnoses were confirmed by standard immunocytochemical labeling of formalin-fixed, paraffin-embedded sections, though it required several days to obtain the results. Direct immunofluorescence using fluorescently conjugated primary antibodies is a practical and rapid method for deciding whether a neurosurgical tumor is a primary glial or an epithelial metastatic tumor in origin. It is the first reported application of the technique for this aspect of rapid neurosurgical diagnosis.     

Comparison of three types of reagents for detection of Bacteroides fragilis by direct immunofluorescence

Three different methods were used to prepare conjugates for the detection of rods of the Bacteroides fragilis group by direct immunofluorescence. Lyophilized conjugates were prepared. Three sets (five in each) of monovalent conjugates against serotype strains of B. fragilis (including conjugate E/E1 + E2) and polyvalent conjugate (A + B + C + D + E1 + E2) were obtained. Each conjugate was prepared in two variants: 1. unabsorbed, 2. absorbed with tissue powder prior to lyophilization. Conjugates obtained by precipitation of sera with 50% ethanol and direct coupling of gammaglobulins with stain were found to meet the requirement for good fluorescence reagents and are well suited for the detection of B. fragilis by direct immunofluorescence. Absorption of the conjugates with tissue powder before lyophilization did not affect their quality.     

Immunotyping of Chlamydia psittaci by indirect immunofluorescence antibody test with monoclonal antibodies

Monoclonal antibodies against pigeon and budgerigar strains of Chlamydia psittaci were used to classify the immunotypes of C. psittaci strains by an indirect immunofluorescence antibody (IFA) test. Thirty-three C. psittaci strains from pigeons and 24 from budgerigars were divided into three immunotypes (P-I, P-II, and P-III) and (B-I, B-II, and B-III), respectively. Two strains from human psittacosis patients were identified as P-III and B-I, coinciding with the epidemiological evidence of each human infection. Two strains from psittacine birds, a parrot and a parakeet, were identical to the B-II immunotype. Other mammalian strains were quite distinct from avian strains in their IFA reaction with the monoclonal antibodies.     

Standardization of factors that influence human urine metabolomics

In nutritional metabolomics a large inter- and intra-subject variability exists, and thus, it becomes important to limit the variance introduced by external factors. In a composite controlled study with full provision of all food for the standardized intervention, human urinary metabolite profiles were investigated for different factors, such as handling of urine collections, diet standardization, diet culture, cohabitation and gender. In study A, 8 healthy subjects (4 men; 4 women) collected 24-h urine, splitting each void into two specimens stored either at 4°C or at room temperature. In study B, 16 healthy subjects (7 men; 9 women) collected 24-h urine for three days while being on a standardized diet. Samples were analyzed by ¹H NMR and chemometrics. The NMR profiles indicated the presence of metabolites presumably originating from bacterial contamination in 3 out of 16 sample collections stored at room temperature. On the contrary, no changes in the NMR profiles due to contamination occurred in the 24-h urine samples stored at 4°C. The study also showed a trend towards a reduced inter- and intra-individual variation during 3 days of diet standardization. In study A, the urine metabolome showed a clear effect of diet culture and cohabitation, but these effects significantly attenuated after diet standardization (study B). Besides, gender-specific differences were found in both studies. Our results emphasize that best practice for any metabolomic study is a standardized, chilled sample collection procedure, and recommend that diet standardization is performed prior to dietary interventions in order to reduce intra- and inter-subject variability.     

Control of propranolol intake by direct chromatographic detection of alpha-naphthoxylactic acid in urine

A rapid chromatographic procedure with a C18 column, a mobile phase of 0.15 M sodium dodecyl sulfate (SDS)-10% (v/v) 1-propanol at pH 3 (0.01 M phosphate buffer), and fluorimetric detection, is reported for the control of propranolol (PPL) intake in urine samples, which are injected directly without any other treatment than filtration. The peak of PPL was only observed in samples taken a few hours after ingestion of the drug due to its extensive conjugation and metabolisation. The detection of several unconjugated PPL metabolites was therefore considered: desisopropylpropranolol (DIP), propranolol glycol (PPG), alpha-naphthoxylactic acid (NLT) and alpha-naphthoxyacetic acid (NAC). NLT showed the best characteristics: it eluted at a much shorter retention time than PPL, its concentration in urine samples was greater and it did not present any interference from endogeneous compounds in urine, common drugs or drugs administered in combination with PPL. The limit of quantification, measured as the concentration of analyte providing a relative standard deviation of 20%, was 24 ng/ml, and the day-to-day imprecision was below 4% for concentrations above 200 ng/ml. The procedure allows the routine control of PPL at therapeutic urine levels. Urinary excretion studies showed that the detection of NLT is possible at least up to 20-30 h after oral administration.     

Detection of Pneumocystis carinii DNA by polymerase chain reaction compared to direct microscopy and immunofluorescence.

The polymerase chain reaction (PCR) using published primers and probes has been compared to conventional stains and immunofluorescence for diagnosis of Pneumocystis carinii. We have screened 71 bronchoalveolar lavage (BAL) fluids from HIV-immunosuppressed patients. Of 34 samples negative by conventional stains and immunofluorescence, only one was positive by PCR. Thirty of 35 samples positive by conventional stains and immunofluorescence were also positive by PCR. One BAL sample, negative by conventional stains but positive by immunofluorescence, was negative by PCR. These data are discussed in relation to clinical and therapeutic conditions of the patients.