Enzyme-linked immunosorbent assay for detection of serum antibody to Blastocystis hominis in symptomatic infections.

An enzyme-linked immunosorbent assay was devised in order to search for antibodies against Blastocystis hominis in infected humans. Reaction proteins were obtained from washed, axenic B. hominis cells, as sonicate. Sonicate was diluted to provide 17 and 34 micrograms of protein per well. Dilutions of patients' sera were applied, followed by phosphatase-conjugated goat anti-human serum and phosphatase substrate. Color was measured at 405 microns wavelength. Immunoglobulin G antibodies to high titers were found. Of 30 sera tested from 28 patients, 3 were negative at the 1/50 threshold dilution, 8 were positive at 1/50, 3 at 1/100, 2 at 1/200, 3 at 1/400, 6 at 1/800, and 5 at 1/1,600. Normal sera (42 blood bank sera) were all negative at 1/50. Each serum was subjected to multiple testing. Duplicate tests were included for each run, and runs were made from 4 to 6 for each serum. Blastocystis hominis is increasingly recognized to be a cause of human enteric disease, with symptoms often like those in giardiasis. Demonstration of strong antibody response is consistent with this view.

Lipid biosynthesis by axenic strains of Blastocystis hominis.

Axenic strains of Blastocystis hominis incorporated 32P, added to the medium as orthophosphate, into a number of phospholipids, including sphingomyelin, cardiolipin, phosphatidic acid, the phosphoglycerides of choline, ethanolamine, serine, and inositol and some other minor phospholipids. Radioactive palmitate and glycerol provided in the growth medium introduced radiolabel into diacylglycerols, triacylglycerols, and all major phosphoglycerides found in the organism. Palmitate is a major fatty acid of cholesterol esters in B. hominis, but radioactive palmitate did not enter the cholesterol ester pool. Radioactive acetate was not incorporated into any lipids. Cholesterol and cholesterol esters of the organism were not labeled when cells were grown in the presence of radioactive glucose, mevalonic acid, or mevalonolactone. Radioactive cholesterol added to the medium became stably associated with B. hominis cells, but none of the radioactive cholesterol entered the cholesterol ester pool. Cholesterol-[3H]-palmitate added to the medium became stably associated with the organism, and most of the radioactivity associated with the cells remained in the cholesterol ester fraction on extended incubation. These results show that this parasitic protozoan has the capacity to synthesize most cellular lipids de novo, but suggest that it acquires free cholesterol and intact cholesterol esters directly from growth medium.

Detection of microsporidian spores in clinical samples by indirect fluorescent-antibody assay using whole-cell antisera to Encephalitozoon cuniculi and Encephalitozoon hellem.

Three polyclonal mouse antisera, to Encephalitozoon cuniculi, Nosema algerae, and Nosema corneum, and two polyclonal rabbit antisera, to E. cuniculi and Encephalitozoon hellem, were used in an indirect fluorescent-antibody assay (IFA) with Enterocytozoon bieneusi, E. cuniculi, and Encephalitgozoon. hellem spores (spores of the last two were taken from culture). Enterocytozoon bieneusi cannot be cultured. By IFA, antisera to E. cuniculi and E. hellem reacted strongly and equally with each other's spores. The mouse antisera reacted strongly with the homologous species, but for these there was segmental and particulate or "dot" staining of heterologous microsporidian spores, indicating cross-reactions with more selected antigens. In fecal samples, cross-reactions with both mouse and rabbit antisera were sometimes seen with different yeast species, with species of streptococci, and species of gram-negative rods. There were no cross-reactions to staphylococci. Enterocytozoon bieneusi was easily identified in duodenal and colonic biopsies, duodenal and colonic fluids, and feces of symptomatic AIDS patients by IFA. In a study of 12 AIDS patients with diarrhea, the new IFA identified microsporidia in all of 11 fecal samples, three colon fluids, six duodenal fluids, and three duodenal biopsy touch preparations. Although the fecal sample of 1 of the 12 was negative, the patient's duodenal fluid contained microsporidian spores by IFA.

Comparative analysis of lipid composition in axenic strains of Blastocystis hominis.

1. Six axenic strains of Blastocystis hominis varied in content of lipids from 12 to 43 pg total lipid/cell. With all strains, phospholipid content was about 39% of total lipids. 2. Neutral lipid fractions of B. hominis were resolved into nine constituents, of which seven were identified tentatively. Sterol esters, principally esters of cholesterol, were the major neutral lipid constituent, accounting for 49-63% of the neutral lipids, and at least 30% of the total lipids. 3. Polar lipids were resolved into eleven constituents, of which nine were identified tentatively. Phosphatidylcholine was the major polar lipid constituent of all strains, accounting for 53-63% of the polar lipids, and about 22% of the total lipids.

Light-microscopic morphology, ultrastructure, culture, and relationship to disease of the nutritional and cell-wall-deficient alpha-hemolytic streptococci.

alpha-Hemolytic streptococci, variously described as cell-wall deficient (C), L form (L), thiol dependent (O), satelliting (S), pyridoxal dependent (PY), and nutritionally deficient (N), or CLOSPYN, were isolated from patients with endocarditis, brain abscess, subauricular abscess, septicemia, acute and chronic urethritis, recurrent aphthous stomatitis, and fever of undetermined origin. With the aid of satelliting, most of the strains were adapted to grow on a human Mycoplasma growth agar consisting of brain-heart infusion agar fortified with 20% human blood, yeast extract, and arginine. Selected CLOSPYN strains required extensive subculture for only partial reversion to parentallike characteristics. Four of six strains biochemically tested were judged Streptococcus morbillorum. Two were unidentifiable. The CLOSPYN form was relatively inert biochemically, but glucose was converted mainly to lactic acid, with acetic acid also present. Guanine-cytosine values were 39%-43%. Cell wall material was present by transmission electron microscopy (TEM), but its synthesis was uneven on single cells and abnormally thickened on other cells. Closely spaced, incompleted septa occurred in cell chains, which resulted in unusually long chains of flattened cells resembling on TEM a stack of checkers. Mesosomes were frequent, greatly enlarged, convoluted, and elongated. They were often sectioned as circular and laminated, with 2-5 layers. Mesosomes were in close contact with nucleoid bodies, which, in turn, were closely apposed or integral with the cytoplasmic membranes in areas of cross-wall development. Chaotic morphology typifies the group. The inclusion of urinary tract infections is new in the gamut of diseases caused by CLOSPYN streptococci.

Phage pattern-specific oxacillin-resistant and borderline oxacillin-resistant Staphylococcus aureus in U.S. hospitals: epidemiological significance.

For a 13-year period (1978 through 1990), oxacillin-resistant (MIC, greater than 4 micrograms/ml) Staphylococcus aureus (ORSA) strains were collected from Clinical Center (National Institutes of Health) patients and patients from five other U.S. hospitals. From Clinical Center patients, 251 of 253 isolates (99%) were bacteriophage typed as phage group III. Five other hospitals contributed 203 ORSA strains, of which 188 (93%) were group III. The group III ORSA strains predominantly included a characteristic core pattern of phages, 7/47/53/54/75/77. For the low-level (borderline) oxacillin-resistant strains (MIC, 2 to 4 micrograms/ml), amoxicillin-clavulanic acid combination (Augmentin) testing disclosed 62 hyper-beta-lactamase producers, of which 59 (95%) were of a separate, distinct S. aureus strain, with the phage pattern 92/94/96/292/D-11 (group V). Thus, ORSA and hyper-beta-lactamase producing S. aureus are distinct epidemic strains.

Blastocystis hominis--past and future.

The history of B. hominis is unique. Few infectious agents have provoked the many misconceptions that plague this enigmatic parasitic ameba. Conflicting descriptions of its nature and pathogenesis have continued throughout the 20th century. As seen by the greatly expanded number of reports in recent years, B. hominis is now a major subject of study, particularly for evidence of disease causation. Physicians are treating patients with intestinal disease caused by B. hominis. Many mild cases resolve in about 3 days without treatment, but others are acute and chronic disease is common. As with E. histolytica, the carrier state is often seen without symptoms. Treatment is usually with metronidazole, but emetine (for refractory infections), trimethoprim-sulfamethoxazole, and pentamidine are also effective. In fecal samples, this complex protozoan appears in a variety of cell forms which makes microscopic diagnosis difficult. As yet, no specific fluorescent-antibody test is available for diagnosis. A culture method to demonstrate the more easily recognized CB form is available, but probably not feasible for most diagnostic laboratories. The common cell forms are the CB form, the granular (mitochondria) form, and the ameba form. The unexpected size range of these forms in clinical material, from yeast size (ca. 7 microns) to giant cells of 20 to 40 microns, makes diagnosis difficult Pseudopodia may be demonstrated by the ameba form in heated microscope stage culture chambers. The anaerobic B. hominis has no cyst form. Its mitochondria are uniquely anaerobic and have no cytochrome protein or oxidative mitochondrial enzymes. Because of its many cell forms and anaerobic mitochondria, B. hominis is an organism of great interest for morphologic and biochemical study. Reproduction is asexual, usually by binary fission. Shizogony occurs in cultured cells. The CB appears to be an organelle whose specific purpose is for reproduction by shizogony. From 2 to 30 progeny are derived from schizogony. The ameba form reproduces by plasmotomy; it has no CB. The pathology of B. hominis infections has been studied in gnotobiotic guinea pigs in which inflammation of the intestinal mucosa and invasion of the superficial layers were seen. Only limited studies of human pathology are available. Those who have studied mucosal histopathology report inflammation and cellular changes that resolve after treatment. More study in this area is strongly indicated (32, 44, 57, 62, 67, 75). Ultrastructural details of B. hominis major forms, except for the schizont, are complete. The organism has no cell wall. The concentric CB takes up as much as 95% of the cell. The major organelles, which include multiple nuclei, Golgi apparatus, mitochondria, endoplasmic reticulum, fat, and other inclusions, are confined in two or four opposed pods in a thin band of peripheral cytoplasm between the spherical entire plasma membrane and the CB membrane. The pods buldge the CB membrane inward. There is evidence of a bacteroid endosymbiont. Education about B. hominis is needed. Entry of recent findings into new textbooks is imperative for its understanding among medical practitioners. Laboratory workers need to be aware of it for many reasons. The College of American Pathologists includes B. hominis in its proficiency testing samples and requires that it be reported from clinical samples.

Magainin analogs effective against pathogenic protozoa.

The in vitro activities of magainin analogs against Blastocystis hominis, Entamoeba histolytica, and Trypanosoma cruzi were assessed by protozoan morphological integrity and motility. The antiprotozoan activities in descending order were magainin B greater than G greater than H, the same order as the alpha-helix contents of the analogs. Magainin B and G were effective against B. hominis, T. cruzi, and E. histolytica.

Stabilities of lyophilized Staphylococcus aureus typing bacteriophages.

Staphylococcus aureus bacteriophages (25 phages) were lyophilized in aliquots 12 to 18 years ago and stored in vacuo at -20 degrees C. Eight viruses each lost one log titer, while seventeen retained the original titers. The use of lyophilized phages provided more reproducible phage typing and reduced by 75% the complexity and cost. This important test is thus made feasible for more laboratories.

Biochemical and ultrastructural study of Blastocystis hominis.

This study was prompted by the paradox of strong presence of mitochondria in an anaerobic protozoan, recently reclassified from the yeasts. Stemming from publication in 1911 to 1912, Blastocystis hominis has been generally accepted as a harmless intestinal yeast of humans, with short standardized textbook (parasitology) descriptions, even to the present day. Reports since 1967 have changed the classification of B. hominis from yeast to protozoan (Sarcodina), and this has been followed by interest in B. hominis-caused disease, resulting in documentation of disease in humans and other primates. In this study of B. hominis, the basic ultrastructure of the mitochondria was shown by thin-section electron microscopy to be identical to that of an archetypical mitochondrion. There were hundreds of them in large B. hominis cells (100 to 200 microns in diameter). Mitochondria were confined to a peripheral ring of cytoplasm bounded by the outer cell membrane (there is no cell wall) and the membrane of the large, spherical, organelle-free central body that constitutes 75% of the cell's volume. Mitochondria tended to surround the cell's usual two to four nuclei. Rhodamine 123 stained the mitochondria selectively, visualized by fluorescence microscopy. The cell was devoid of cytochromes. Addition of 0.1% cytochrome c to the growth medium increased utilization of glucose by 34% and that of lactate by 17%. Furthermore, it markedly increased the number of mitochondrion-filled cells. At higher concentrations, cytochrome c inhibited the growth of the cells. Despite the presence of large numbers of mitochondria, activities of the mitochondrial enzymes pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, isocitrate dehydrogenase, glutamate dehydrogenase, and cytochrome c oxidase were absent. Thus, the function of the mitochondria in B. hominis remains unknown. Considerable activities of aspartate aminotransferase and alanine aminotransferase were found. Aldolase activity was prominent. Pyruvate decarboxylase was present. Diaphorase and lactate dehydrogenase were detectable but in suspect quantities. Other missing enzymes were gamma glutamyl transpeptidase, alkaline phosphatase (a lysosomal marker), and creatine kinase isoenzymes.

Cyniclomyces guttulatus (Saccharomycopsis guttulata)--culture, ultrastructure and physiology.

Organisms that form an essential extra inner lining of selected areas of the stomach mucosa occur in mice, rats and some other animals. The yeast Cyniclomyces guttulatus (Saccharomycopsis guttulata) was shown in this study to line the stomach of domestic and feral rabbits, guinea pigs, and chinchillas. The layer of yeast cells formed a loose barrier between lumen contents and mucosal surface. A rapid rate of multiplication in the stomach provided yeast cells that blended in with stomach lumen contents, passed through the gut, and were finally excreted in large numbers in fecal pellets. Ascospore formation occurred during passage through the large intestine. The layer of yeast cells lining the stomach had no evident salubrious nor deleterious effect on the animal. C. guttulatus grew rapidly from stomach contents or single fecal pellets in a new enriched semisolid medium. Growth was good at pH 1 through 8 on the solidified enriched medium. A very unusual characteristic of C. guttulatus is optimal growth at 38 degrees C, and growth at 42 degrees C, with failure to grow below 30 degrees C. TEM demonstrated a very thick, laminated cell wall which had a thick, filamentous external coating. There were mitochondria, polyribosomes, lipid droplets, and an unusually large central nucleus. The developing spore nucleus became extremely electron dense and encapsulated, along with condensed mitochondria, ribosomes, short membrane sections and other organelles, in a dense lamellar covering.

Blastocystis hominis, a long-misunderstood intestinal parasite.

For over 50 years, Blastocystis hominis has been held to be a harmless intestinal yeast-probably frequent in stool samples from man and other primates, but usually ignored except as a possible source of confusion with Entamoeba histolytica. More recently, its status as a protozoan parasite has been accepted, and it is now increasingly recognized as an agent of intestinal disease - usually self-limiting but occasionally fatal in monkeys. Here, Charles Zierdt reviews the status o f this intriguing protozoan, drawing attention to its unusual biochemistry.

Colonization of newly arrived house staff by virulent staphylococcal phage types endemic to a hospital environment.

The acquisition of hospital strains of Staphylococcus aureus by new house officers was studied in an 800-bed referral hospital over a 1-year period. S. aureus isolates, including three strains with characteristic phage patterns that had previously been documented to cause disease in patients and colonize hospital personnel, were recovered from the anterior nares of 35 of 54 newly arrived house officers. There was a significant correlation (r = 0.7475; P less than 0.02) between colonization with the dominant hospital strain (S) and exposure to the hospital environment over 12 months. No hospital-wide increase in infections owing to the S strain was seen during this period, which suggests that house staff acquired this strain from reservoirs within the hospital. The finding of colonization with virulent endemic S. aureus strains in house officers working on every ward of the hospital suggests that new strategies for control of S. aureus nosocomial infections must be considered and evaluated.

Simplified lysed-blood culture technique.

A blood culture system was developed in which a lysing agent (either Tween 20, one of several other polyoxyethylene adducts, digitonin, or Triton X-100) is added to the blood culture medium. Of 33 Triton compounds, 9 lysed human blood, as did 7 of 21 polyoxyethylene compounds and digitonin, all at a concentration of 0.05%. Under the specific test conditions, three of the hemolytic polyoxyethylene compounds and digitonin had no inhibitory effect. All of the Triton compounds had at least some inhibitory effect on the most sensitive of the pathogenic bacteria that were tested, Streptococcus pneumoniae and Neisseria meningitidis. Because of results from previous studies, Triton X-100 was tested further, despite evidence in this study of its inhibition of bacteria. Of the 55 lysing agents tested, digitonin, Triton X-100, Brij 96, and Tween 20 were selected for further testing as additions to conventional culture broth. Comparative culture studies with bacteremic blood from infected rabbits were performed with the conventional blood culture, the Isolator system (Du Pont Co., Wilmington, Del.), and the new lysing medium. The new system has the advantages of lysis filtration and lysis centrifugation without the associated added cost and processing complexity.

Cytochrome-free mitochondria of an anaerobic protozoan--Blastocystis hominis.

Isolated mitochondria of the anaerobic protozoan Blastocystis hominis were subjected to spectral analysis, color, catalase, and peroxidase tests and found to be completely negative for cytochrome enzymes, catalase, and peroxide. Based on the absence of cytochrome enzymes, the possible evolution of B. hominis mitochondria from anaerobic bacteria is postulated.