Development of specific fluorescent-antibody test for tissue form of Penicillium marneffei.

The diagnosis of penicilliosis marneffei can be difficult because the clinical manifestations mimic those of tuberculosis, histoplasmosis, and other mycotic infections. Furthermore, the tissue form of Penicillium marneffei can be confused with those of Histoplasma capsulatum and Cryptococcus neoformans. To facilitate the rapid detection and identification of P. marneffei in clinical materials, we sought to develop a specific indirect fluorescent-antibody (IFA) reagent for this dimorphic pathogen. Preliminary IFA studies with yeast-like cells (fission arthroconidia) of P. marneffei indicated that these cellular elements stained with antiglobulins against culture filtrate antigens and whole yeast-like cellular antigens. Both types of antiglobulins reacted with the yeast-like cells of P. marneffei and with H. capsulatum, but not with their respective mycelial forms. The antiglobulins also failed to react with the yeast and hyphal forms of a variety of other heterologous fungi. Specific antiglobulins useful in an IFA test for identifying P. marneffei yeast-like cells in culture or in clinical materials were produced by adsorptions with yeast-form cells of H. capsulatum. The yeast-like culture filtrate antigens of P. marneffei are preferred for use in the production of the specific antiglobulins because they stained P. marneffei yeast-like elements more intensely than antiglobulins produced against intact yeast-like cells.

A tissue culture bilayer model to study the passage of Neisseria meningitidis.

A tissue culture bilayer system has been developed as a model to study the mechanisms of attachment and invasion involved in the pathogenesis of Neisseria meningitidis. The model incorporates epithelial and endothelial cell layers separated by a microporous membrane and makes it possible to observe and quantify the passage of bacteria through the multiple layers and to study the mechanisms by which they make this passage. This model is adaptable to a wide variety of microbial pathogens and can be modified by substituting any physiologically relevant eucaryotic cells for the component layers. The system's makeup of cells of human origin and its reproducibility give it advantages over animal and primary organ culture models, while the added complexity of multiple layers allowing cell-to-cell communication makes it a more realistic human tissue model than standard cell monolayers.

Morphologic and staining characteristics of a cyanobacterium-like organism associated with diarrhea.

A spherical organism 9-10 microns in diameter, seen in three outbreaks of diarrhea in Southeast Asia and the United States during the past 2 years, bore characteristics of a cyanobacterium when observed in formalin-preserved stool specimens and by electron microscopy. Organisms in freshly passed stool specimens showed an internal morula of lipid-containing globules. In fresh water, the morula divided into two sausage-shaped structures resembling the sporocysts of an isosporid coccidian. After 7 months, the organisms had not developed the crescentic sporozoites seen in the Coccidia but had begun to multiply slowly in culture. It was impossible to stain the internal structures of the organisms because the outer cyst wall ruptured during desiccation, releasing the contents of the cysts. The organisms were readily identified by their intense blue autofluorescence under UV light, but they were also recognizable by bright-field microscopy and by a modified acid-fast stain. Almost all infected persons suffered intermittent diarrhea for 2-3 weeks and many emphasized a feeling of intense fatigue during the course of their illness.

Specific immunofluorescence staining of Treponema pallidum in smears and tissues.

To date, tissue sections prepared from Formalin-fixed tissues have not been successfully stained with Treponema pallidum subspecies-specific antibody in a direct fluorescent-antibody assay. While current methods stain T. pallidum, they do not distinguish T. pallidum from other spirochetes such as Borrelia burgdorferi (E. F. Hunter, P. W. Greer, B. L. Swisher, A. R. Simons, C. E. Farshy, J. A. Crawford, and K. R. Sulzer, Arch. Pathol. Lab. Med. 108:878-880, 1984). Because trypsin pretreatment of tissue sections has enhanced other immunofluorescent-antibody (IFA) applications, we compared the use of the trypsin digestion method with the current 1% ammonium hydroxide (NH4OH) method as a means to obtain specific staining of T. pallidum in tissues by both direct and indirect IFA techniques. Pretreated T. pallidum-infected tissues sections from rabbits, hamsters, and humans were quantitatively examined with the direct fluorescent-antibody-T. pallidum test conjugate absorbed with Treponema phagedenis, the Reiter treponeme. For indirect staining, a serum specimen from a patients with syphilis absorbed by affinity chromatography with T. phagedenis was used as the primary reagent, and a fluorescein isothiocyanate-labeled rabbit anti-human globulin was used as the secondary reagent. Serum specificity was established first by examining antigen smears of T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, B. burgdorferi, T. phagedenis, and Treponema denticola MRB and then by examining tissues infected with these pathogens plus those infected with four Leptospira serovars. When we stained tissue using the direct IFA method that is currently a standard method for the examination of chancre smears, we found it to be unsuitable for use with tissue. Trypsin digestion did not offer an improvement over the NH4OH pretreatment method in the specific identification of T. pallidum by direct IFA. However, specific identification of T. pallidum in tissue sections was obtained by the indirect IFA technique after either trypsin or NH4OH pretreatment.

Comparative study with in situ hybridization and immunocytochemistry in detection of HIV-1 in formalin-fixed paraffin-embedded cell cultures.

The sensitivities of three immunohistological techniques were compared in this study for detecting human immunodeficiency virus (HIV-1) in infected cultured human lymphocytes that had been formalin-fixed and paraffin-embedded. The techniques included in situ hybridization (ISH) with HIV-1 cDNA; immunocytochemistry with HIV-1 p24 monoclonal antibody (ICC-m); and immunocytochemistry with HIV-1 polyclonal antibody from a patient with acquired immunodeficiency syndrome (AIDS) (ICC-p). Procedures were optimized for enzyme digestion and for antibody reaction conditions. HIV-1--infected cells and noninfected control cells were tested. Noninfected controls were uniformally negative by all three methods. Infected cells had the highest positivity rate by the ISH method (p less than or equal to 0.0001), and the ICC-p method was more positive than the ICC-m (p less than or equal to 0.0001). Both the ICC-p and the ICC-m techniques were more positive with the cocultivated cell cultures than the ISH, which was more sensitive with the infected continuous cell line (P less than or equal to 0.0001). The ICC-p method had a lower standard deviation on positive results than either the ICC-m or ISH method. The variability observed with these test procedures, reagents, and specimens suggests that these are important technological parameters in detecting p24, with implications for detecting other HIV-1 markers in infected tissues.

Immunofluorescent staining of Treponema in tissues fixed with formalin.

Immunofluorescent examination of formalin-fixed tissue for Treponema pallidum has generally been unsatisfactory because of nonspecific background fluorescence and poor contrast. We examined the process of treating deparaffinized formalin-fixed tissue sections with 1% ammonium hydroxide (NH4OH) to improve fluorescent staining. Treponema pallidum- and Treponema pertenue-infected rabbit testes or human tissue biopsy specimens fixed in 10% buffered formalin and embedded in paraffin were examined. Sections were cut one week to five years after embedment. Tissues were then stained with fluorescein- or rhodamine-labeled human anti- T pallidum globulin for 30 minutes at 37 degrees C. Treponemes were consistently stained and background staining was generally reduced after NH4OH treatment in both fresh and stored tissue. Cutting sections at a thickness of approximately 2 micron was critical to achieve optimal fluorescence.