Phyllis Gyorkey

A morphologic study on the occurrence and distribution of structures resembling viral nucleocapsids in collagen diseases

Abstract Cytoplasmic filamentous structures resembling unenveloped nucleocapsids of paramyxo- or related viruses were found in vascular endothelial cells of the kidney and skin and in circulating leukocytes from patients with systemic lupus erythematosus (SLE) (fifty-two cases) and systemic sclerosis (four cases). These structures also occurred in the synovium in rheumatoid arthritis (four cases), in the vascular endothelial cells of the glomeruli in Sjogrens syndrome (three cases) and in the muscle cells in polymyositis (three cases). The structures were most abundant in systemic lupus erythematosus. It is proposed that the presence of these structures in large numbers suggests the diagnosis of systemic lupus erythematosus. These structures were absent in patients with procainamideinduced lupus syndrome and in eighty-three patients with kidney diseases other than those of the connective (collagen) tissues. These structures were also absent in nineteen patients without kidney disease or collagen disease. Occasionally, similar structures were found in one patient who received a renal transplant and in two patients with sickle cell disease; in these cases the extreme rarity of the structures was conspicuous. Morphologic studies suggest that the structures undergo developmental phases comparable to those of myxoviruses. The pathogenesis of collagen diseases is compatible with the etiologic role of a defective virus.

The Role of Microsporidia in the Pathogenesis of HIV-Related Chronic Diarrhea

A common problem in persons infected with the human immunodeficiency virus (HIV) is chronic diarrhea, which is reported to occur in approximately 50% of patients with the acquired immunodeficiency syndrome (AIDS) [1]. In many cases, diarrhea can be attributed to infection with one or more common (for example, Salmonella species, Giardia lamblia) or opportunistic (for example, Cryptosporidium species) enteric pathogens [2]; however, in one half of patients with AIDS and diarrhea, no enteric pathogen can be identified despite intensive investigations [3, 4]. The two main hypotheses proposed to account for these cases of unexplained chronic diarrhea are direct mucosal HIV infection [5] and infection with one or more currently unidentified or unrecognized enteric organisms. An emerging enteric pathogen is the intracellular protozoan Microsporidia [3, 4, 6], a well-known cause of infection in animals [6-8]. Gastrointestinal microsporidians were first observed in humans in 1985 in electron microscopic studies of small-bowel biopsy specimens from two patients with AIDS who had unexplained chronic diarrhea [9, 10]. In subsequent studies of duodenal and jejunal biopsy specimens, the microsporidian Enterocytozoon bieneusi was detected in as many as 30% of HIV-infected patients with unexplained chronic diarrhea [3]; in one study [11], the organism was detected only in patients with severe defects in cellular immunity (defined by CD4 cell counts 100 cells/mm3). In our ongoing studies of the pathogenesis of HIV-related chronic diarrhea, we have conducted a systematic electron microscopic examination of endoscopic mucosal biopsy specimens obtained from HIV-infected men with and without chronic diarrhea. In the course of this work, we detected E. bieneusi in the duodenal tissue obtained from men without diarrhea, which is contrary to previous findings. The purpose of the study we now report was to determine whether microsporidiosis is associated with HIV-related chronic diarrhea. Methods Study Sample In April 1991, we began a casecontrol study of HIV-infected patients who were enrolled if they had at least one recent (within 1 month) negative stool culture for bacterial pathogens and one negative stool examination for ova and parasites. Cases included patients with chronic diarrhea that was either continuous (defined by the Centers for Disease Control and Prevention as two or more loose stools per day for 30 days [12]) or intermittent (defined as episodes of two or more loose stools per day alternating with episodes of formed stools for 30 days). Patients without diarrhea served as controls. The patients were recruited from the Houston Veterans Affairs Medical Center Special Medicine Clinic, an outpatient facility that provides comprehensive care to approximately 500 veterans with HIV infection. The patients in our study were recruited directly by the investigators or were referred by the clinic staff. We sought to enroll 100 patients, including an equal number of unmatched cases and controls. The study was designed with 50 patients in each group (1- = 80%, = 0.05) because we expected the proportion of controls with microsporidiosis to be low (that is, less than 20%). With 50 participants in each group, we expected to be able to detect clinically meaningful differences (30% or greater) in the proportions of cases and controls with microsporidiosis, allowing us to find statistical significance with an odds ratio of 4.0 or greater. The study was approved by the institutional review boards for human subjects at the Houston Veterans Affairs Medical Center and Baylor College of Medicine. Clinical Data and CD4 Cell Count Each patient in the study underwent a history and physical examination to determine the route of HIV exposure (subsequently referred to as transmission category) and to collect demographic, clinical (previous AIDS-defining diagnoses, stool frequency, stool consistency), and therapeutic data (treatment with zidovudine or trimethoprim-sulfamethoxazole). For all participants, a CD4 cell count was obtained within 1 month of recruitment. The information was recorded on a data collection form designed specifically for the project. Microbiologic Studies At the time of endoscopy, a repeated fresh stool specimen was obtained for bacterial culture and examination for ova and parasites. One mucosal biopsy specimen from each site (duodenum, sigmoid colon, rectum) was cultured for Mycobacterium species. Endoscopy and Biopsy Esophagogastroduodenoscopy and flexible sigmoidoscopy were done using Fujinon videoendoscopes (Fujinon Inc., Wayne, New Jersey). Biopsy specimens, taken with Jumbo forceps, were obtained at three sites: the distal duodenum, the sigmoid colon (25 to 60 cm from the anal verge), and the rectum (12 cm from the anal verge). Light Microscopy Three biopsy specimens from each site [duodenum, sigmoid colon, rectum] were fixed in 10% buffered formalin and embedded in paraffin. Serial 4-msections were cut, deposited on slides, and stained with 1) hematoxylin and eosin for the evaluation of the mucosal morphology and the detection of Microsporidia, Cryptosporidium species, and viral inclusions; 2) Ziehl-Neelsen for the detection of acid-fast bacteria such as Mycobacterium species; and 3) Giemsa and tissue Gram stain for the detection of Microsporidia. The histopathologist was initially blinded; after the electron microscopy results were available, the biopsy specimens were re-examined in an attempt to identify the organisms. Electron Microscopy One biopsy specimen from each site (duodenum, sigmoid colon, rectum) was fixed in phosphate-buffered 2% glutaraldehyde, postfixed in 1% phosphate-buffered osmium tetroxide, embedded in plastic resin, fine-sectioned on LKB 8800 or Reichert-Jung SUPER NOVA Ultramicrotomes, stained with uranyl acetate and lead citrate, and examined under the Philips 301 and JEOL 1200 electron microscopes. The pathologist-electron microscopist was blinded to the patients clinical and laboratory data when the biopsy specimens were examined and the diagnosis was rendered. Data Analysis Data were entered into a computer database (Paradox, Borland International, Scotts Valley, California). Statistical analyses were done using STATA software (Computing Resource Center, Santa Monica, California). Differences in the mean age and CD4 cell count and in the distribution of variables (transmission category, intensity of infection) were assessed using the t-test and chi-square statistic, respectively. An overall odds ratio for having microsporidiosis was calculated; 95% CIs were calculated using the Woolf method [13]. Odds ratios adjusted for CD4 cell count and the presence of other enteric pathogens were calculated using the Mantel-Haenszel procedure and logistic regression. Results Characteristics of the Study Sample Between 1 April 1991 and 30 November 1992, we enrolled 106 HIV-infected men, 55 with chronic diarrhea and 51 without diarrhea. The clinical characteristics of the patients are summarized in Table 1. The mean age of all participants was 43 years, and the mean age of patients in the two groups was similar. The transmission categories were as follows: homosexual or bisexual men with or without a history of intravenous drug use (95 of 106 [90%]); and heterosexual men with or without a history of intravenous drug use (11 of 106 [10%]). The proportion of homosexual or bisexual men among the cases and controls was similar (Table 1). The mean CD4 cell count of all study participants was 151 cells/mm3 (range, 1 to 900 cells/mm3); the mean CD4 cell counts were significantly different (P = 0.02) in cases (113 cells/mm3) and controls (193 cells/mm3) (normal range, 517 to 1677 cells/mm3). Table 1. Clinical Characteristics of the Patients* Presence of Other Enteric Pathogens In the overall study sample, enteric pathogens other than Microsporidia were identified in 24 of 106 men (23%). The proportions of cases and controls with other enteric pathogens were 29% (16 of 55) and 16% (8 of 51), respectively (P = 0.10) (Table 1). These organisms were detected in stool specimens in 9 men (Giardia lamblia in 4, E. histolytica in 3, Shigella and Campylobacter species in 1 each); light microscopy showed organisms in an additional 10 men (cytomegalovirus in 5, acid-fast bacilli in 4, and both organisms in 1); electron microscopy showed cytomegalovirus in an additional man; and mycobacterial culture of biopsy tissue showed Mycobacterium avium complex in an additional four men. Microsporidia Infection Prevalence and Sites of Infection Electron microscopic examination of duodenal biopsy specimens showed E. bieneusi infection in 31 of 106 men (29%). Organisms were detected in specimens from the sigmoid colon in 3 of these 31 men, and 1 of these 3 men also had organisms in a biopsy specimen from the rectum. The electron micrographs in Figures 1 and 2 show the characteristic features [14] of E. bieneusi observed in the duodenal biopsy specimens. Figure 1. Electron graph of early proliferative plasmodia (thin arrows) and later sporogonial plasmodium (thick arrow) of Enterocytozoon bieneusi in a duodenal biopsy specimen. Figure 2. Electron graph of mature spores (arrows) of Enterocytozoon bieneusi in a duodenal biopsy specimen. Light Microscopy Compared with Electron Microscopy for Detecting Microsporidia At the initial light microscopic examination, Microsporidia were not detected. However, after the electron microscopy results were available, the biopsy specimens of the 31 men with microsporidiosis were re-examined. Sections stained with hematoxylin and eosin revealed lightly stained oval bodies in the villous tips [3] in 11 of the 31 men. In 2 men, rare aggregates of small refractile supranuclear bodies, presumably spores, were identified. No Microsporidia were detected in sections stained with Giemsa or Churukian tissue Gram stain [15]. No Microsporidia were detected in sections from the sigmoid colon and rectum. The propor

The usefulness of electron microscopy in the diagnosis of human tumors

Ultrastructural features of neoplastic cells can provide clues for correct diagnosis when light microscopy fails. Secretory granules are characteristic in the following tumors: mucin granules in poorly differentiated adenocarcinomas, zymogen granules in acinic cell carcinomas, lysosomal granules in prostatic carcinomas, melanin granules in malignant melanoma, carcinoid, islet cell tumors, pheochromocytoma, and neuroblastoma granules in the corresponding neoplasms. Among cytoplasmic organelles, rough surfaced endoplasmic reticulum characterizes adrenocortical, ovarian, and hepatocellular carcinomas and plasmacytomas. Tonofibrils are characteristic of squamous cell carcinomas. Glycogen deposits distinguish Ewings sarcoma from lymphoreticular neoplasms. Intercellular relationships and membrane specialization are important features in the differential diagnosis of various undifferentiated tumors. The frequent resolution of difficult diagnostic problems by electron microscopy outweighs the disadvantages of this technique, such as the expense and time required.

Electron microscopic observations on structures resembling myxovirus in human sarcomas

Human tumors of mesenchymal origin contain cytoplasmic structures resembling ribonucleoprotein strands of paramyxoviruses. Similar structures have previously been reported in collagen diseases. The nature and function of these structures remain unresolved.

Hairy Cells in Long-Term Cultures

Excerpt To the editor: The derivation of hairy cells is unsettled because arguments can be put forward for both monocytic and lymphocytic origins. In a recent report (1), lymphocytic derivation of ...