Use of electron microscopy in examination of faeces and rectal and jejunal biopsy specimens.

The stools and rectal biopsy specimens of 44 patients with AIDS and diarrhoea were examined by culture, light microscopy, and electron microscopy. In 13 patients examination of rectal biopsy material and faecal samples showed no pathogen, but in two of these, microsporidiosis was found by electron microscopical examination of jejunal biopsy specimens. This organism was also identified electron microscopically in one of the further five jejunal biopsy samples taken from patients with a known cause of diarrhoea. Blastocystis hominis infection was identified electron microscopically in six patients, all of whom had cryptosporidiosis additionally seen by light microscopy. Four of these six patients remained well for long periods, with only moderate diarrhoea, and follow up showed no evidence of blastocystis infection. In only four of 11 patients found to have cryptosporidium in their stools at light microscopy were organisms found at electron microscopy. Viral inclusions were only identified at electron microscopy in one of 10 patients with an opportunistic viral infection seen at light microscopy (cytomegalovirus n = 7, herpes simplex virus n = 3). No additional viral pathogens were detected in either stools or rectal biopsy material by electron microscopy. It is concluded that routine electron microscopic examination of stool samples or rectal biopsy material taken from patients with AIDS and diarrhoea is unnecessary and does not increase the yield of potential pathogens compared with standard microbiological techniques and histology.     

Cytomegalovirus infection in gastrointestinal tracts of patients infected with HIV-1 or AIDS.

All gastrointestinal tract biopsy specimens from 190 patients positive for HIV-1 or with AIDS were reviewed to assess the prevalence of cytomegalovirus (CMV) infection, morphology of infected cells, and the associated histopathological features. Eighteen patients (10 (7.7%) of 129 HIV antibody positive and eight (13.1%) of 61 with AIDS) had CMV identified in 35 biopsy specimens from the following sites: oesophagus (n = 3); stomach (n = 6); small intestine (n = 4); colorectum (n = 18) and perianal area (n = 4). Eleven patients had CMV alone as the potential cause of symptoms and in seven there were coexistent pathogens or Kaposi's sarcoma. The appearance and type of infected cells at different sites was highly variable. Immunocytochemical techniques and electron microscopic examination were performed to confirm the presence of CMV antigen and CMV virus particles and to exclude the possibility of an adenovirus producing similar cytopathic changes. It is important to recognise the different morphological forms of infected cells, and the use of immunocytochemical techniques is recommended in patients at risk for CMV or in whom CMV infection is suspected.     

Histological diagnosis of intestinal microsporidiosis in patients with AIDS.

Fifty nine patients seropositive for human immunodeficiency virus (HIV) and diarrhoea and 20 with weight loss were investigated for microsporidiosis using light and electron microscopical examination of duodenal and jejunal biopsy specimens. Eight cases of microsporidiosis were found, in five of whom it was the sole pathogen. In all eight cases the organism was identified at light microscopy without prior knowledge of the electron microscopical findings. All stages of the life cycle are best seen in resin sections cut at 1 micron and stained with Giemsa, but spores could easily be identified in paraffin sections cut at 5 microns and stained with haematoxylin and eosin. In all cases the parasite was identified both in duodenal pinch and jejunal "Crosby" capsule biopsy specimens. All cases of microsporidiosis occurred in patients with diarrhoea. Both electron and light microscopical examination suggested that the pathogenic mechanism involves the shedding of infected enterocytes containing large numbers of spores. It is suggested that the optimal way to diagnose microsporidiosis is by light microscopical examination of duodenal pinch biopsy specimens.     

Detection of microsporidia (Enterocytozoon bieneusi) in intestinal biopsy specimens from human immunodeficiency virus-infected patients by PCR.

Intestinal microsporidiosis has been implicated as a major cause of chronic diarrhea in human immunodeficiency virus (HIV)-infected patients. So far diagnosis depends on direct visualization of the parasites by light and transmission electron microscopy. We evaluated the diagnostic value of microsporidian DNA amplification by PCR on duodenal biopsy specimens obtained from patients with and without intestinal microsporidiosis caused by Enterocytozoon bieneusi. Thirteen HIV-infected patients (all CDC stage C3) were studied. Eight patients had intestinal microsporidiosis caused by E. bieneusi (n = 6), Septata intestinalis (n = 1), and Encephalitozoon cuniculi (n = 1); microsporidioses were diagnosed by light microscopy of stool samples and confirmed by light and electron microscopy of intestinal biopsy specimens. Five patients had no microsporidia in their stool samples or in their intestinal biopsy specimens, as examined by light and electron microscopy. Additionally, DNA prepared from Toxoplasma gondii derived from mouse ascites was used as a further control. A 353-bp DNA fragment of the small-subunit rRNA gene could be amplified from all six biopsy specimens infected with E. bieneusi, and the nature of the PCR products was confirmed by Southern blot hybridization. No amplification of DNA fragments was seen by using DNA extracted from biopsy specimens with S. intestinalis or E. cuniculi infection or without microsporidian infection and with template DNA extracted from T. gondii. The results suggest that PCR testing of intestinal biopsy specimens may be a useful approach to diagnosing microsporidiosis in HIV-infected patients.     

Diagnosis of intestinal and disseminated microsporidial infections in patients with HIV by a new rapid fluorescence technique.

AIMS--To assess the value of a new rapid fluorescence method for the diagnosis of microsporidiosis in HIV seropositive patients. METHODS--Microsporidian spores in stools were demonstrated by using the fluorochrome stain Uvitex 2B. The new technique was evaluated in three groups of HIV seropositive patients with diarrhoea. Group 1: 19 patients with biopsy confirmed E bieneusi infection (186 stool samples); group 2: 143 consecutive patients from whom faeces were submitted for routine investigation of diarrhoea (318 samples); group 3: 16 patients with small intestinal biopsy specimens negative for microsporidia (55 samples). The new method was used to monitor spore shedding during experimental treatment with paromomycin and albendazole in four patients. RESULTS--Brightly fluorescent spores were detected in all stool samples of patients in group 1. In group 2 16 (11%) patients had spores in their stool samples. E bieneusi was found in 11 patients; in the other five another genus of microsporidia, Encephalitozoon, was recognised. Encephalitozoon spores were also found in the urine of three of these patients and in the maxillary sinus aspirate of two of them, suggesting disseminated infection. The results were confirmed by electron microscopic examination. In group 3 negative biopsy specimens were confirmed by negative stool samples in all cases. Treatment with albendazole and paromomycin did not affect the spore shedding in three patients with E bieneusi infection. By contrast, in a patient with Encephalitozoon sp infection albendazole treatment resulted in clinical improvement together with complete cessation of spore excretion in the stool. CONCLUSION--The Uvitex 2B fluorescence method combines speed, sensitivity, and specificity for the diagnosis and treatment evaluation of intestinal and disseminated microsporidiosis.     

Human polyoma virus in renal allograft biopsies: morphological findings and correlation with urine cytology.

Human polyoma virus (PV) interstitial nephritis occurs in immunosuppressed patients after reactivation of latent virus in renal epithelium. Currently, there is neither general consensus about the incidence of clinically significant PV infection in renal transplants nor conclusive evidence determining its significance in the long-term graft outcome. We evaluated 601 renal transplant biopsy specimens (from 365 patients) by routine light microscopy and immunoperoxidase stains with antibody against SV40 (which cross reacts with PV). We also examined urine samples from 200 patients (100 obtained concurrently with a renal biopsy in patients presenting with acute graft dysfunction and 100 from patients with stable graft function). Electron microscopic evaluation was performed in 50 renal biopsy specimens and in 23% of all urine samples. PV was identified in 1.8% biopsy specimens (1.9% of patients). PV interstitial nephritis showed the typical viral cytopathic changes in tubular epithelial cells associated with marked tubular damage and a disproportionately mild degree of tubulitis. There was no difference in the incidence of PV in the urine of patients with acutely deteriorating versus stable renal function (18% and 19%, respectively); however, urines with large numbers of infected cells (> 10/cytospin) and inflammatory changes in the sediments corresponded invariably to patients with acute allograft dysfunction (8 of 8), and in most cases to biopsy specimens showing PV interstitial nephritis (7 of 8). Based on these findings, urine samples seem to be the most sensitive and cost-effective screening method for PV infection; only urine samples with inflamed sediments and abundant infected cells correlate with clinically significant disease. In these cases, examination of a renal biopsy is indicated. Immunohistochemical stains are useful to confirm the presence of PV but do not increase the sensitivity of diagnosis of PV if this is not already suspected on routine light microscopy. In our material, immunostains were helpful ruling out the presence of PV in a small number of biopsy specimens (2%) that showed markedly reactive tubular cells resembling PV infection. Most patients with PV interstitial nephritis responded to decreased immunosuppression; however, the decay in graft function (based on creatinine slopes) was significantly more rapid in these patients than in matched controls. Evidence of PV infection should be systematically sought in renal biopsy specimens and urine samples from renal allograft recipients.     

Opportunistic infections with coronavirus-like particles in patients infected with the human immunodeficiency virus?

From 35 patients infected with Human Immunodeficiency Virus (HIV) and belonging to various risk groups, 60 stool specimens were examined for the presence of Coronavirus-like particles (CVLP) using electron microscopy. CVLP were detected in 5 (8%) stool samples from 5 different patients (14%). Only one of the patients had diarrhoea. The five patients with CVLP-positive stools were all at advanced stages of HIV infection. The remarkable discrepancy between our data and another study, reporting a rate of 50% CVLP-positive HIV patients, most of them persistently shedding the virus, is discussed.     

Human immunodeficiency virus and papovavirus infections in acquired immunodeficiency syndrome: an ultrastructural study of three cases

A wide variety of neurologic conditions associated with the acquired immunodeficiency syndrome (AIDS) have been attributed to human immunodeficiency virus (HIV) infection of the central nervous system (CNS). Tissue samples from the brains of three patients with AIDS, diagnosed as having CNS toxoplasmosis on the basis of computed tomographic scans of the head, were studied by transmission electron microscopy. In two, HIV particles were observed budding from, in close association with, and in cytoplasmic vacuoles of mononuclear and multinucleated macrophages, but no other cell types. The patient with the greatest number of HIV particles also had large amounts of papovavirus (progressive multifocal leukoencephalopathy) in the nuclei of oligodendroglial cells and in the cytoplasm of astrocytes. These astrocytes often had atypical features at the light microscopic level. Following an initial biopsy that showed only HIV, primary CNS lymphoma was diagnosed by needle biopsy and confirmed at autopsy in a second case. A diagnosis of progressive multifocal leukoencephalopathy was rendered by transmission electron microscopy in a third case, but no HIV was detected. Toxoplasmosis was not confirmed in any of the three cases. Diagnosis of CNS lesions in patients with AIDS should not rely exclusively on radiography but include biopsy for both light and transmission electron microscopy. Transmission electron microscopy can be employed to reveal HIV and papovavirus infections not discernible at the light microscopic level and should be used as a diagnostic tool in HIV-related infections.     

A powerful DNA extraction method and PCR for detection of microsporidia in clinical stool specimens

The diagnosis of intestinal microsporidiosis has traditionally depended on direct visualization of the parasite in stool specimens or intestinal biopsy samples by light and/or electron microscopy. Limited information about the specificity and sensitivity of PCR for the detection microsporidia in clinical stool specimens is available. To establish a sensitive and specific method for the detection of microsporidia in clinical samples, we studied clinical stool specimens of 104 randomly selected human immunodeficiency virus-infected patients with diarrhea to compare light microscopy and PCR. Fluorochrome Uvitex 2B staining was used for light microscopy. To raise the sensitivity of PCR, we used a powerful and fast DNA extraction method including stool sedimentation, glass bead disruption, and proteinase K and chitinase digestion. PCR was performed with primer pairs V1-PMP2, V1-EB450, and V1-SI500, and the nature of the PCR products was confirmed by Southern blot hybridization. Microsporidiosis was diagnosed by light microscopy in eight patients. Ten patients tested positive for microsporidiosis by PCR. Enterocytozoon bieneusi was found in seven cases, and Encephalitozoon intestinalis was found in four cases. In one case a double infection with E. bieneusi and E. intestinalis was diagnosed by PCR, whereas light microscopy showed only E. bieneusi infection. PCR testing of stool specimens is useful for diagnosis and species differentiation of intestinal microsporidiosis in HIV patients.     

The use of electron microscopy for the diagnosis of cancer in bronchial biopsies

To investigate whether electron microscopic examination of bronchial biopsy specimens was of any additional value in the diagnosis of lung cancer, we examined 235 consecutive biopsy specimens embedded in Epon. Semithin sections were used to establish a light microscopic diagnosis, and the results were compared with those of electron microscopy. Ninety-six biopsies contained tumor. In 13 instances, the light microscopic diagnosis had to be revised after electron microscopic examination. Another 5 biopsies contained tissue suspected to be tumor; in 1 biopsy this possibility could be ruled out. Three biopsies contained tumor suspected of being small cell cancer, and this diagnosis was confirmed by electron microscopy. Electron microscopy was helpful in 17 of 106 biopsies. Histopathologic examination of surgically resected material from 18 patients confirmed the electron microscopic results of biopsies. We conclude that electron microscopy of bronchial biopsy specimens gives important additional information for accurate diagnosis. For practical purposes, however, specimens should be embedded in Epon or glycol-methacrylate for light microscopy. Developments in immunohistologic techniques will change choices of histologic techniques.     

A Powerful DNA Extraction Method and PCR for Detection of Microsporidia in Clinical Stool Specimens

The diagnosis of intestinal microsporidiosis has traditionally depended on direct visualization of the parasite in stool specimens or intestinal biopsy samples by light and/or electron microscopy. Limited information about the specificity and sensitivity of PCR for the detection microsporidia in clinical stool specimens is available. To establish a sensitive and specific method for the detection of microsporidia in clinical samples, we studied clinical stool specimens of 104 randomly selected human immunodeficiency virus-infected patients with diarrhea to compare light microscopy and PCR. Fluorochrome Uvitex 2B staining was used for light microscopy. To raise the sensitivity of PCR, we used a powerful and fast DNA extraction method including stool sedimentation, glass bead disruption, and proteinase K and chitinase digestion. PCR was performed with primer pairs V1-PMP2, V1-EB450, and V1-SI500, and the nature of the PCR products was confirmed by Southern blot hybridization. Microsporidiosis was diagnosed by light microscopy in eight patients. Ten patients tested positive for microsporidiosis by PCR. Enterocytozoon bieneusi was found in seven cases, and Encephalitozoon intestinalis was found in four cases. In one case a double infection with E. bieneusi and E. intestinalis was diagnosed by PCR, whereas light microscopy showed only E. bieneusi infection. PCR testing of stool specimens is useful for diagnosis and species differentiation of intestinal microsporidiosis in HIV patients.     

Pathology of astrovirus associated diarrhoea in a paediatric bone marrow transplant recipient

Human astrovirus infection often causes outbreaks of self limiting diarrhoea, but may also infect patients who are immunodeficient or immunocompromised. Although there are previous publications relating to various aspects of astroviruses, there is a minimal amount of literature on the histopathological features of gastrointestinal astrovirus infection in humans. We report the histopathological findings, including immunohistochemical and electron microscopic features, of astrovirus infection in a bone marrow transplant recipient aged 4 years with diarrhoea. The appearance of a small intestinal biopsy did not suggest graft versus host disease, but demonstrated villous blunting, irregularity of surface epithelial cells, and an increase in lamina propria inflammatory cell density. Immunohistochemical staining with a murine astrovirus group specific monoclonal antibody demonstrated progressively more extensive staining in the duodenal and jejunal biopsies, predominantly restricted to the luminal surface and cytoplasm of surface epithelial cells, most marked at the villus tips. Electron microscopic examination demonstrated viral particles within the cytoplasm of enterocytes, focally forming paracrystalline arrays.     

Visualization of the ribosome-lamella complex in plastic-embedded biopsy specimens as an aid to diagnosis of hairy-cell leukemia.

Hairy-cell leukemia is a lymphoid leukemia of B-cell lineage, the cells of which are characteristically tartrate resistant acid phosphatase positive on blood and bone marrow smears. However, because hairy-cell leukemia is frequently associated with abundant marrow stroma, dry marrow taps, and peripheral pancytopenia, the diagnosis may rest on the appearance of the bone marrow biopsy specimen alone. The ribosome-lamella complex has been associated with hairy-cell leukemia, and can be visualized by light microscopy using l-micron sections of plastic-embedded bone marrow specimens stained with toluidine blue. We describe the findings in a case in which bone marrow and liver biopsy specimens were positive for hairy cells containing ribosome-lamella complex, which were visualized with both electron microscopy and light microscopy. Reliable light microscopic identification of ribosome-lamella complex may provide an easy and inexpensive method of aiding in the diagnosis of hairy-cell leukemia when aspirate material is not available for tartrate-resistant acid phosphatase stain.     

The ultrastructure of small cell lung carcinoma in bronchial biopsy specimens

Forty-three bronchial biopsy specimens from patients with small cell lung cancer (SCLC) were studied by electron microscopy. In 38 specimens the diagnosis was based on the light microscopic examination of Epon-embedded tissue; 36 of these specimens contained dense-core granules on electron microscopic examination. In five cases the light microscopic diagnosis was either different from the electron microscopic diagnosis or in doubt. Electron microscopy revealed dense-core granules as the only sign of differentiation, and the diagnosis was changed to SCLC. The tumor cell populations in the biopsy specimens were quite heterogeneous. Cells of the oat cell type were always present and, on electron microscopic examination, always showed degenerative changes. It was therefore decided that this cell type represents an artifact. The true SCLC tumor cell, which constitutes only a small portion of the tumor in biopsy specimens, is characterized by a regular oval or rounded cell with pale cytoplasm and a ground-glass nucleus with finely dispersed chromatin. Nucleolated cells, similar to those seen in large cell cancer, are often present but are not ultrastructurally different from nonnucleolated tumor cells.     

Toxoplasmosis of donor and recipient hearts after heterotopic cardiac transplantation

Toxoplasmosis of both donor and recipient hearts was diagnosed by means of endomyocardial biopsy specimens after heterotopic cardiac transplantation for dilated cardiomyopathy. Before transplantation, the donor had raised antibody titers to Toxoplasma, and the recipient was negative. When toxoplasmosis was diagnosed on the basis of endomyocardial biopsy specimen, the recipient had a greatly elevated antibody titer of 1:1,027. This suggests that the infection could have been transferred with the donor heart. The mononuclear cell response elicited by disrupted toxoplasmic cysts interferes with the diagnosis of rejection in graft biopsy specimens. Electron microscopy is valuable in confirming a light microscopic diagnosis of toxoplasmosis. Drug therapy eradicated the toxoplasmosis, but the patient died later of tuberculous meningitis.     

Shorter survival in HIV-positive patients with diarrhoea who excrete adenovirus from the GI tract.

Adenoviruses have been described as a cause of diarrhoea in patients infected with the human immunodeficiency virus (HIV). The prevalence of adenoviruses was studied in all HIV-positive patients presenting with diarrhoea at the Royal Free Hospital in London between 1991 and 1995. In addition, all postmortems carried out in HIV-positive individuals registered at the same centre between 1990 and 1997 were reviewed for evidence of adenovirus infection. Adenovirus was detected in 16.1% of patients presenting with diarrhoea. These individuals had a significantly lower CD4 count and were more likely to have had a diagnosis of acquired immunodeficiency syndrome (AIDS) than patients with diarrhoea in whom adenovirus was not detected. The median survival was 1 year compared with 2.4 years for those without adenoviruses; this difference remained significant (P = .008) after controlling for differences in CD4 counts between the groups. Gastrointestinal adenovirus excretion occurs at an advanced stage of HIV disease, and is associated with a poor prognosis. We suggest that adenoviruses may contribute to mortality in this population.     

Role of Electron Microscopy in Transplant Renal Pathology

The crucial role that electron microscopy plays in diagnostic renal pathology is undisputed. By allowing recognition of findings not identifiable by light microscopic evaluation, electron microscopy has contributed significantly to the understanding of renal diseases and has proven to be of unquestionable value in many diagnostic situations. However, the percentage of cases in which electron microscopic examination adds important information that is either key for establishing or confirming a diagnosis or provides valuable data that influence patient's management remains controversial. This figure depends on the renal biopsy service that is surveyed, but it is reported that on the average ultrastructural evaluation is of value in approximately 30 to 45% of the cases. Correct interpretation of a renal biopsy depends on the ability to correlate light, immunofluorescence, and ultrastructural findings. In contrast, the role of electron microscopy in the examination of renal transplant specimens remains controversial. Many centers do not use routine electron microscopy to examine these specimens and insist that there are only a few specific indications that require ultrastructural evaluation. There is general agreement among renal pathologists that electron microscopy is of importance in the evaluation of renal specimens from patients with proteinuria to distinguish between transplant glomerulopathy, recurrent or de novo glomerulonephritis in order to correctly manage these patients and predict survival of the graft. The other possible indications are much more controversial. This paper summarizes and critically reviews the literature available on this subject and defines recommendations based on the information available at the current time.     

Rapid diagnosis of infectious bursal disease infection by immunofluorescence on clinical material

Direct immunofluorescence on impression smears of bursa of Fabricius and direct electron microscopic examination of bursal specimens were compared with virus isolation in chick embryo fibroblasts (CEF) and embryonated eggs for the diagnosis of infectious bursal disease (IBD). In chicks experimentally infected with a virulent strain of virus, immunofluorescence was a more sensitive method of demonstrating infection than direct electron microscopy or virus isolation. In chicks experimentally infected with an avirulent strain of virus, immunofluorescence and virus isolation in CEF were equally sensitive methods of demonstrating infection and more sensitive than direct electron microscopy or virus isolation in embryonated eggs. In two field surveys immunofluorescence was, overall, more sensitive than virus isolation and direct electron microscopy and gave a good correlation with histopathological diagnosis of IBD.     

Intestinal microsporidiosis as a cause of diarrhea in human immunodeficiency virus-infected patients: a report of 20 cases.

Chronic diarrhea accompanied by weight loss is a common and often debilitating problem associated with human immunodeficiency virus (HIV) infection. Enterocytozoon bieneusi, a newly identified species of the phylum of protozoa, Microspora, has been reported associated with chronic diarrhea and wasting in 11 acquired immunodeficiency syndrome (AIDS) patients in the United States, Europe, and Africa. Diagnosis has been based solely on the ultrastructural identification of this small, intracellular parasite in bowel biopsies. Seventy-one small bowel biopsies from 67 homosexual AIDS and AIDS-related complex patients with chronic diarrhea and with no pathogens identified by light microscopy on paraffin sections, were embedded in plastic and studied by light and transmission electron microscopy. Enterocytozoon bieneusi microsporidiosis was diagnosed by electron microscopy in 20 (22 biopsies) of the patients. More jejunal biopsies (16 of 36) were positive than duodenal biopsies (six of 35). Parasites and spores were clearly visible at the light microscopic level in the semi-thin plastic sections from 17 and 21 of the biopsies, respectively. In retrospect, parasites could be identified by light microscopy in standard hematoxylin and eosin-stained paraffin sections. Infection was confined to enterocytes covering the villi, especially the tips, and was associated with villous atrophy and cell degeneration, necrosis, and sloughing. Release of spores into the bowel lumen was evident. Colorectal biopsies from two of the patients with small bowel microsporidiosis were negative for microsporidia. Enterocytozoon bieneusi infection of the small bowel may be an important cause of diarrhea in HIV-infected persons.