Detection of cytomegalovirus DNA in classic and epidemic Kaposi's sarcoma by in situ hybridization

Several lines of evidence have suggested an etiologic association of cytomegalovirus (CMV) with Kaposi's sarcoma. This contention is supported by a pathoepidemiologic survey of 54 cases of acquired immunodeficiency syndrome (AIDS) at our own institution. Of the 27 patients with documented Kaposi's sarcoma, 24 (89%) showed histologic evidence of CMV infection (cytomegalic cells with viral inclusions), whereas only 9 (33%) of the patients with AIDS without Kaposi's sarcoma showed hallmarks of CMV infection. In an attempt to address this question further, we have searched for the presence of CMV nucleic acid sequences in a series of 25 patients with AIDS and Kaposi's sarcoma, using the technique of in situ DNA hybridization. The reliability of the in situ technique is demonstrated, and the technique is shown to be more sensitive than the detection of viral inclusions within Kaposi's sarcoma lesions by routine light microscopy. However, only 20% of our cases showed evidence of CMV involvement, and the CMV-positive cells within the affected Kaposi's sarcoma lesions were few and sparsely distributed. In addition, a companion series of 6 elderly patients with "classic" Kaposi's sarcoma showed no evidence of CMV infection by either conventional microscopy or in situ hybridization. These results do not support the notion of a strong association between Kaposi's sarcoma and CMV, unless the CMV sequences are present at a copy number too low for detection by these methods. The implications of these findings in light of current theories of CMV oncogenesis are discussed.     

Comparison of immunoperoxidase and DNA in situ hybridization techniques in the diagnosis of cytomegalovirus colitis

Cytomegalovirus (CMV) infection of the colon occurs in immunocompromised patients and in patients with ulcerative colitis. In the former, it can cause serious bleeding or colonic perforation and in the latter, it may precipitate a fulminant phase of illness. The authors compared the immunoperoxidase technique with a monoclonal antibody (Mab) against CMV early antigen and DNA in situ hybridization using a biotinylated probe in the identification of virus-infected cells in colon samples from patients with known CMV colitis and with fulminant ulcerative colitis without histologic evidence of infection. The Mab stained the nuclei of infected cells without recognizable viral inclusions particularly in cases showing few characteristic viral inclusions. In well-developed CMV infections, with many inclusions, immunoperoxidase staining was less prominent. The biotinylated DNA probe recognized cytopathic cells with prominent inclusions but was only rarely positive in smaller cells. In specimens with few inclusions, there was no staining with in situ hybridization. No positivity was observed by either technique in cases of fulminant ulcerative colitis with no morphologic evidence of CMV infection. The authors conclude that immunoperoxidase using monoclonal anti-CMV early antigen adds useful information in the evaluation of early or focal cases of CMV colitis. DNA in situ hybridization is usually positive in cytopathic cells and is less useful for diagnosis. CMV could not be demonstrated in cases of fulminant ulcerative colitis when viral inclusions were not observed in routine preparations.     

Invasive amoebiasis in two patients with AIDS and cytomegalovirus colitis.

Homosexual persons or human immunodeficiency virus (HIV)-infected patients frequently excrete cysts of nonpathogenic strains of Entamoeba histolytica ("Entamoeba dispar"). However, invasive amoebiasis is rare. We report two patients with AIDS and cytomegalovirus colitis in whom invasive amoebiasis was histologically diagnosed. It is concluded that E. histolytica has to be considered in HIV-infected patients with colitis.     

In situ hybridization for cytomegalovirus DNA in AIDS patients.

Infection by cytomegalovirus (CMV) is a frequent cause of morbidity and mortality in patients with acquired immune deficiency syndrome (AIDS). The authors studied the distribution of CMV in 4 patients with AIDS using a commercially available, biotin-labeled CMV DNA probe for in situ hybridization and immunohistochemical staining for the detection of CMV antigen in formalin-fixed paraffin-embedded tissues. The sensitivity and specificity of the hybridization procedure was demonstrated by appropriate controls. The immunohistochemical test for the detection of CMV antigen in routine histologic sections was less sensitive than the in situ hybridization method. CMV DNA was detected not only in cytomegalic inclusion cells, but also in nuclei and cytoplasm of histologically normal-appearing cells such as endothelial cells, pneumocytes, hepatocytes, biliary epithelium, gastrointestinal epithelium, Langerhans islet cells, acinar and duct epithelium of pancreas, adrenal cortical and medullary cells, and prostate epithelium. In addition, CMV DNA, but not CMV antigen, was found in polymorphonuclear leukocytes. These cells may serve as intermediate host or reservoir of CMV and may transmit posttransfusion CMV infection. In situ hybridization on routine histologic sections with a biotinylated CMV DNA probe is a rapid, sensitive, and specific method for diagnostic and experimental pathology.     

The ultrastructural and immunohistochemical demonstration of viral particles in lymph nodes from human immunodeficiency virus-related and non-human immunodeficiency virus-related lymphadenopathy syndromes

Viral particles have been demonstrated by electron microscopy in lymph nodes from patients with acquired immune deficiency syndrome AIDS-related persistent generalized lymphadenopathy (PGL) syndrome. Immunohistochemical and in situ hybridization studies have identified these viruses as the human immunodeficiency virus (HIV). In this study, we examined 20 PGL lymph nodes and found viral particles in 18 cases. Immunohistochemical studies on these cases revealed positive staining for the HIV core protein P24 within germinal centers of secondary follicles. In addition we found viral particles, morphologically indistinguishable from those observed in PGL lymph nodes, in 13 of 15 non-HIV related reactive lymph nodes. Immunohistochemical staining of these lymph nodes for the P24 core protein was negative. None of the patients in this group had risk factors for developing AIDS and none exhibited clinical evidence of immune deficiency. We conclude that the viral particles observed in PGL lymph nodes are most likely HIV, but similar particles can be seen in reactive lymph nodes not associated with HIV infection. The discrete localization of these particles within germinal centers has been observed for other viruses and immune complexes and a possible mechanism of this antigen deposition is discussed.     

Transsynaptic spread of varicella zoster virus through the visual system: a mechanism of viral dissemination in the central nervous system

We report a patient with pathologic evidence of anterograde spread of varicella zoster virus (VZV) through the visual system. A 29-year-old homosexual man developed the acquired immunodeficiency syndrome (AIDS) 2 months before the onset of left herpes zoster ophthalmicus. During the next 11 months, the zoster infection progressed to involve the left eye, with resultant keratitis, iritis, retinitis, and eventual blindness. Later, the patient developed bilateral blindness, left hemiparesis, and fatal pneumonia. At autopsy, the brain revealed destruction of the visual system and adjacent structures, with sparing of the remainder of the brain. Glial cells near the areas of necrosis showed Cowdry type A intranuclear inclusions. In situ hybridization with probes to VZV nucleic acid sequences were positive in the necrotic brain and retinal areas. Hybridization with probes to cytomegalovirus, herpes simplex virus type II, human immunodeficiency virus, and Epstein-Barr virus were negative. Electron microscopy revealed characteristic herpes group nucleocapsids. This case provides insight into the mechanisms of virus dissemination and the production of encephalitis.     

Pathological Features of Virus Infections of the Central Nervous System (CNS) in the Acquired Immunodeficiency Syndrome (AIDS)

Neurological disorders are a common cause of morbidity and mortality in the acquired immunodeficiency syndrome (AIDS). In this report we describe the neuropathological changes associated with both human immunodeficiency virus (HIV) infection and with the major opportunistic virus infections, cytomegalovirus (CMV), JC papovavirus (JCV) and herpes simplex virus (HSV) seen in AIDS. In addition "in situ" hybridization studies have been employed for the detection of virus genomic material in each case and the usefulness of this method in supporting the pathological diagnosis is demonstrated. Mechanisms whereby HIV infection results in leukoencephaiopathy and the possible contributing roles of the three opportunistic virus infections are discussed.     

Pathological features of virus infections of the central nervous system (CNS) in the acquired immunodeficiency syndrome (AIDS).

Neurological disorders are a common cause of morbidity and mortality in the acquired immunodeficiency syndrome (AIDS). In this report we describe the neuropathological changes associated with both human immunodeficiency virus (HIV) infection and with the major opportunistic virus infections, cytomegalovirus (CMV), JC papovavirus (JCV) and herpes simplex virus (HSV) seen in AIDS. In addition "in situ" hybridization studies have been employed for the detection of virus genomic material in each case and the usefulness of this method in supporting the pathological diagnosis is demonstrated. Mechanisms whereby HIV infection results in leukoencephalopathy and the possible contributing roles of the three opportunistic virus infections are discussed.     

Elevated levels of CD4 antigen in sera of human immunodeficiency virus-infected populations.

CD4 antigen levels in sera from asymptomatic intravenous drug users and homosexuals and patients with lymphadenopathy, acquired immunodeficiency syndrome-related complex, or acquired immunodeficiency syndrome were quantitated. Like soluble CD8, CD4 antigen levels were elevated in human immunodeficiency virus-seronegative asymptomatic intravenous drug users and homosexuals, probably reflecting infections such as cytomegalovirus, Epstein-Barr virus, and hepatitis B virus infections. The sera from human immunodeficiency virus-seropositive groups of patients with human immunodeficiency virus infection also had elevated levels of CD4 antigen, presumably reflecting infections like cytomegalovirus and human immunodeficiency virus infections.     

Diagnostic pathology in the acquired immunodeficiency syndrome. Surgical pathology and cytology experience with 67 patients

We report the pathologic findings in specimens submitted for histologic and cytologic evaluation from 67 patients with the acquired immunodeficiency syndrome. A wide variety of opportunistic pathogens were identified in 41 patients. Mycobacterium avium-intracellulare evoked only a mild host response: granulomas, if present, were poorly formed. Biopsy specimens showing cytomegalovirus gastroenteritis required sections at multiple levels to demonstrate inclusions. Combined histologic and cytologic evaluation can increase the diagnostic yield in pulmonary and esophageal infections. Kaposi's sarcoma was found in biopsy specimens from 29 patients. Early lesions were often extremely subtle, yet distinct from, benign vascular proliferations in involuted lymph nodes. Malignant lymphoma was diagnosed in ten homosexual men who were suspected of having the acquired immunodeficiency syndrome. The lymphomas were characterized by B-cell origin, a diffuse pattern, frequent extranodal presentations, and an aggressive clinical course with prominent central nervous system involvement.     

Cytomegalovirus vasculitis and colon perforation in a patient with the acquired immunodeficiency syndrome

We describe a patient with the acquired immunodeficiency syndrome who suffered a colon perforation which we believe was directly attributable to disseminated cytomegalovirus (CMV) infection. Thrombosed vessels within the submucosa and muscle wall contained evidence of CMV vasculitis, while adjacent vessels without viral inclusions were fully patent. This report supports other evidence that CMV may act as a primary etiologic agent of gastrointestinal disease, particularly in the immunocompromised host. The increased recognition of CMV as a cause of significant morbidity in certain gastrointestinal lesions becomes especially important with the advent of newer antiviral therapy specifically directed against CMV infection.     

Elevated soluble CD8 levels in sera of human immunodeficiency virus-infected populations

Soluble CD8 levels in sera were quantitated in asymptomatic intravenous drug abusers, homosexuals, and patients with lymphadenopathy or acquired immunodeficiency syndrome. Soluble CD8 levels were elevated in human immunodeficiency virus-seronegative intravenous drug abusers and homosexuals, probably reflecting infections like cytomegalovirus. The sera of human immunodeficiency virus-seropositive groups of patients with human immunodeficiency virus infection also had elevated levels of soluble CD8, reflecting infections like cytomegalovirus and human immunodeficiency virus infection.     

In situ DNA hybridization for cytomegalovirus in colonoscopic biopsies

We reviewed colonoscopic biopsies of the lower gastrointestinal tract performed during a two-year period. Those representing neoplasia were excluded. Formalin-fixed paraffin-embedded biopsy specimens from 53 patients were studied by in situ DNA hybridization for cytomegalovirus (CMV) using commercially available biotinylated DNA probes detected by an avidin-biotin peroxidase technique. Nine of the patients were severely immunocompromised: four had acquired immunodeficiency syndrome, three had ulcerative colitis and were receiving high-dose steroid therapy, one was a bone marrow transplant recipient, and one had idiopathic pulmonary fibrosis and was receiving therapy with prednisone and cyclophosphamide. Four of these had evidence of CMV infection by routine histology and DNA hybridization. Three additional immunocompromised patients had evidence of CMV infection by DNA hybridization alone. Forty-four patients had inflammatory conditions or ulcerations of the lower gastrointestinal tract. Six of these had evidence of CMV by DNA hybridization alone. Histologically normal as well as enlarged and cytomegalic cells were probe positive, and the cells were sparse to numerous in number. They were found in the epithelium and/or lamina propria. This technique was demonstrated to be applicable to routinely processed colonic biopsy specimens.     

Profound CD4+ T lymphocytopenia in human immunodeficiency virus negative individuals,improved with anti-human herpes virus treatment

Lymphocytopenia and CD4+ T lymphocytopenia can be associated with many bacterial, fungal, parasite and viral infections. They can also be found in autoimmune and neoplastic diseases, common variable immunodeficiency syndrome, physical, psychological and traumatic stress, malnutrition and immunosuppressive therapy. Besides, they can also be brought into relation, without a known cause, with idiopathic CD4+ T lymphocytopenia. Among viral infections, the Retrovirus, specially the human immunodeficiency virus, is the most frequently cause. However, many acute viral infections, including cytomegalovirus and Epstein Barr virus can be associated with transient lymphocytopenia and CD4+ T lymphocytopenia. As is well known, transient lymphocytopenia and CD4+ T lymphocytopenia are temporary and overcome when the disease improves. Nonetheless, severe CD4+ T Lymphocytopenia associated with chronic infections by human herpes virus has not been reported. We describe 6 cases of human immunodeficiency virus negative patients, with chronic cytomegalovirus and Epstein Barr virus infections and profound lymphocytopenia with clinical symptoms of cellular immunodeficiency. These patients improved rapidly with ganciclovir or valganciclovir treatment. We claim here that it is important to consider the chronic human herpes virus infection in the differential diagnosis of profoundly CD4+ T lymphocytopenia etiology, when human immunodeficiency virus is absent, in order to start effective treatment and to determine, in future studies, the impact of chronic human herpes virus infection in human beings'' health.     

In situ hybridization AT-tailing with catalyzed signal amplification for sensitive and specific in situ detection of human immunodeficiency virus-1 mRNA in formalin-fixed and paraffin-embedded tissues

In situ hybridization is one of the most important techniques to visualize gene expression at the cellular level in various tissues. The in situ hybridization-AT tailing (ISH-AT) method uses a specially designed and synthesized oligonucleotide probe that has (AT)10 on the 3' side. This (AT)10 of the probe is elongated by DeltaTth DNA polymerase in the presence of dATP, dTTP, and labeled dUTP in the tissue after hybridization. Through this process the target is labeled with many hapten molecules. In this study, we detected human immunodeficiency virus type 1 RNA in formalin-fixed and paraffin-embedded tissues obtained from autopsied patients with acquired immunodeficiency syndrome by combining ISH-AT with the catalyzed signal amplification (CSA) system (ISH-AT-CSA), although we failed to detect signals from the same samples by conventional in situ hybridization using RNA probes (RISH) with CSA (RISH-CSA). We demonstrated that the ISH-AT-CSA method was superior to RISH-CSA in terms of both sensitivity and specificity, and that it was applicable to fluorescence in situ hybridization and double staining with immunohistochemistry for the characterization of cell phenotypes.     

Epstein-Barr viral load as a marker of lymphoma in AIDS patients

Epstein-Barr virus (EBV) is implicated in the pathogenesis of acquired immunodeficiency syndrome (AIDS) lymphoma, and viral DNA is present within the malignant cells in about half of affected patients. We examined the extent to which EBV viral load is elevated in the plasma of AIDS lymphoma patients compared to AIDS patients with opportunistic infections. Sixty-one AIDS patients were studied including 35 with lymphoma (24 non-Hodgkin, six Hodgkin, and five brain lymphoma) and 26 with various opportunistic infections. In situ hybridization revealed EBV encoded RNA (EBER) expression in the malignant cells of 17/28 AIDS lymphomas (61%). In 232 serial plasma samples from 35 lymphoma patients and in 128 samples from AIDS controls, EBV viral load was assayed by quantitative-polymerase chain reaction (Q-PCR) using a TaqMan probe targeting the BamH1W sequence. EBV was detected in plasma from all 17 EBER-positive AIDS lymphoma patients, with viral loads ranging from 34 to 1,500,000 copies per ml (median 3,210). Viral load usually fell rapidly upon initiation of lymphoma therapy and remained undetectable except in two patients with persistent tumor. In 11 AIDS patients, whose lymphoma lacked EBER expression, and in 26 control patients without lymphoma, levels of EBV in plasma were usually low or undetectable (range 0-1,995 and 0-2,409, median 0 and 0, respectively). There was no association between EBV viral load and human immunodeficiency virus (HIV) load or CD4 count. In conclusion, EBV viral load shows promise as a tool to assist in diagnosis and management of EBV-related lymphoma patients.     

The clinicopathological significance of cytomegalovirus inclusions demonstrated by endocervical biopsy.

A healthy 20 yr old woman presented for evaluation following a cervical smear which showed viral effects typical of human papilloma virus. Colposcopy showed changes of cervicitis with the main finding on histologic examination of biopsy material being an acute and chronic cervicitis associated with typical features of cytomegalovirus (CMV) infection. Viral identification was confirmed by immunoperoxidase staining, in situ hybridization and electron microscopy. The patient was lost to follow up for 18 mths. Following this, a repeat colposcopy again showed inflammation, with cervicitis, mild dysplasia and CMV inclusions on biopsy. Full immunological work-up, including human immunodeficiency virus (HIV) study, was performed and was normal. Only 11 other cases of endocervical biopsies with histological evidence of CMV inclusions were found in the literature, although the reported rate of detection of genital CMV in women on culture is 4-12%. In the 9 cases where information was available, endocervical inflammation was present. One patient was on immunosuppressive medication for systemic lupus erythematosus and another was found to have Acquired Immune Deficiency Syndrome (17% of total). These cases demonstrate that although histologic examination is an insensitive marker for CMV within the cervix, its presence may signify immunodeficiency and so immunological assessment of a patient with this finding is advisable.