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.     

Cytomegalovirus infection in classic, endemic and epidemic Kaposi's sarcoma analyzed by in situ hybridization.

In order to evaluate the association between cytomegalovirus (CMV) infection and Kaposi's sarcoma (KS) at the cellular level, four classic KS, five endemic (African) KS and 55 epidemic KS from 12 patients with AIDS were analyzed by in situ hybridization with a biotinylated specific CMV-DNA probe. CMV-DNA was sparsely demonstrated in the sarcomas in 10 of the patients with AIDS but in none of the classic or endemic sarcomas. The distribution and localization of the CMV-infected cells did not suggest CMV as a major pathogenic stimulus for the development of KS but rather as an opportunistic infection in severely immunosuppressed patients.     

Implausibility of an aetiological association between cytomegalovirus and Kaposi''s sarcoma shown by four techniques.

The presence of cytomegalovirus (CMV) was analysed in either lymph node or skin and lung tissue necropsy specimens affected by Kaposi's sarcoma, from 10 patients who had died of AIDS. The different detection techniques used were: (i) immunohistochemical demonstration of CMV immediate early antigen (IEA); (ii) in situ hybridisation with a biotinylated CMV DNA probe; (iii) Southern blot hybridisation of DNA extracted from sequential tissue sections; and (iv) polymerase chain reaction (PCR) with CMV specific primers on the DNA samples. The results of these analyses were compared with the postmortem data on CMV obtained by infectious particle assays and histological examination, especially of adrenal glands of the same patients. The results of the various detection methods correlated very well, yielding a combined score of six of 10 patients positive for CMV; there did not seem to be any association between the presence of CMV and the occurrence of Kaposi's sarcoma in our patients.     

In situ hybridization for the detection of cytomegalovirus (CMV) infection

Summary Five autopsy cases of cytomegalovirus (CMV) infections were studied. Conventional light microscopy disclosed characteristic cytopathic effects in lungs, kidneys, and brain. In one case, electron microscopy was carried out and revealed typical herpesvirus particles.In situ hybridization was done with biotin-labeled CMV-DNA probes and an avidin-alkaline phosphatase detection system. 4/5 cases were observed to contain hybridizing cells in different organs. Intensity of hybridization was related to the severity of CMV infection, roughly estimated by counting cytomegalic cells. In addition to cytomegalic cells, a high number of normal-looking epithelial and mesenchymal cell types were positive. These latter cells showed nuclear hybridizations in contrast to cytomegalic cells which hybridized both within the nuclei and the cell bodies.This modified in situ hybridization procedure is a rapid and valuable tool for the detection and final demonstration of virus infection, and will be of particular help for the examination of paraffin-embedded specimens.Dedicated to Prof. Dr. G. Seifert on the occasion of his 65th birthdayThis study was supported by a grant of the Deutsche Forschungsgemeinschaft (Lo 285/2-3) and the Hamburger Stiftung zur Förderung der Krebsbekämpfung (I 208)     

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.     

Problems in detection of cytomegalovirus in urine samples by dot blot hybridization.

A hybridization assay for the detection of cytomegalovirus (CMV) in urine specimens was established. Two different DNA fragments were used as hybridization probes: the HindIII L fragment (11.7 kilobases) and the EcoRI J fragment (10.6 kilobases) of the human CMV strain AD169. These probes were used in an isolated and highly purified form and therefore did not cross hybridize with vector sequences. As shown by hybridization with DNA from CMV-infected and uninfected cells, the assay was highly CMV specific and sensitive (detection limit, 750 to 500 fg of CMV DNA). A total of 122 urine specimens were examined by DNA hybridization, virus isolation, and the detection of CMV-induced early nuclear protein. The results coincided in 91% of the samples. The application of DNA hybridization to urine samples, however, is not without problems, and some of the pitfalls and drawbacks are discussed.     

Cytomegalovirus, angiomatosis, and Kaposi's sarcoma: new observations of a debated relationship.

Kaposi's sarcoma (KS) encompasses a broad spectrum of lesions ranging from foci of muco-cutaneous angiomatosis to tumor masses of internal organs. Its strong association with immune deficiency and the marked differences in incidence between the various populations at risk are suggestive of an infectious etiology. The agent most often suspected of being implicated in the etiology of KS is cytomegalovirus (CMV); however, despite sustained research on this subject, its role remains controversial. The present work includes six cases of KS with a broad variety of lesions in which, with the use of light and electron microscopy, immunohistochemistry, and in situ hybridization, we investigated the presence of CMV and examined its relationship with KS. CMV was present in all six cases and showed a remarkable propensity for the KS lesions where both intranuclear and intracytoplasmic forms were not only next to but frequently within KS cells. Areas of angiomatosis, hemorrhage, and KS had usually an abundance of CMV. Herpes-like virus particles inside KS nuclei were documented by light and electron microscopy and identified as CMV by immunohistochemistry and in situ hybridization. The selective morphologic presence of this virus within the tumor cells, not previously demonstrated, indicates a strong association between CMV and KS, the significance of which remains to be established.     

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.     

Atypical cytomegalovirus inclusions in gastrointestinal biopsy specimens from patients with the acquired immunodeficiency syndrome: diagnostic role of in situ nucleic acid hybridization.

Cytomegalovirus (CMV) infection of the gastrointestinal tract is a common cause of morbidity and mortality in the acquired immunodeficiency syndrome. The proper recognition of CMV-infected cells in gastrointestinal mucosal biopsies is critical so that effective therapy is not delayed, preventing further viral dissemination. Although the pathology criteria for classic CMV inclusions have been well described, the occurrence of morphologically atypical inclusions has been reported but the inclusions are not well characterized. This study prospectively examined the relative frequency of classic and atypical CMV inclusions in gastrointestinal mucosal biopsy specimens from 13 human immunodeficiency virus-positive symptomatic patients. The results demonstrated that classic inclusions were rarely found, including four esophageal, one gastric, and one colonic biopsy specimens in which none were seen. However, atypical CMV inclusions were identified from all biopsy specimens examined; these inclusions were much more numerous than classic inclusions and could be categorized into three morphologic types. The atypical inclusions were difficult to precisely identify as CMV-infected cells, but in situ DNA hybridization for CMV was valuable in establishing their viral origin, thus permitting the correct etiologic diagnosis.     

Rapid diagnosis of cytomegalovirus encephalitis in patients with AIDS using in situ hybridisation.

AIMS--To evaluate the presence of cytomegalovirus (CMV) DNA in the cerebrospinal fluid of patients with AIDS and suspected viral encephalitis using an in situ hybridisation assay with digoxigenin labelled CMV DNA probes. METHODS--The presence of CMV DNA was evaluated in cerebrospinal fluid cells of 10 patients with AIDS using in situ hybridisation. The positivity of CMV DNA was confirmed by the presence of CMV induced antigens in the same specimens. The presence of CMV DNA and CMV induced antigens was also analysed in peripheral blood leucocytes. The time required to perform the in situ hybridisation assay was about eight hours. RESULTS--The in situ hybridisation assay was sensitive, specific, and provided good resolution. Six patients proved positive for the presence of CMV DNA in CSF cells and all six also proved positive for CMV DNA in blood leucocytes. Of the six CMV positive patients, five were treated with specific antiviral drugs: of these, one died during the treatment while four clinically recovered after one month of treatment. CONCLUSIONS--The in situ hybridisation assay using digoxigenin labelled CMV DNA probes can be used as a valuable diagnostic test for the detection of CMV DNA in the cerebrospinal fluid cells of patients with suspected CMV encephalitis and can therefore prompt adequate antiviral therapeutic intervention.     

Hepatic Cytomegalovirus Involvement in Autopsy Cases

Hepatic involvement of cytomegalovirus (CMV) was studied in 15 autopsy cases with disseminated or solitary CMV infection. Formalin-fixed, paraffin-embedded tissue sections were examined by immunohistochemistry (IHC) using a monoclonal antibody against early and late CMV antigens, and by in situ hybridization (ISH) using biotinylated CMV DNA probes. Three cases showed cytomegalic cells in liver sections by conventional staining, five showed hybridization with CMV DNA probes and seven showed reaction with the monoclonal antibody in the liver. CMV infection was detected not only in cytomegalic cells but also in many non-cytomegalic cells by IHC and ISH, proving these techniques to be superior to routine histological examination. The inflammatory reaction in the liver was not prominent. The reason for the weak inflammatory response in the liver of our present cases, and the possible availability of IHC and ISH for analysis of liver biopsy and bronchoalveolar lavage specimens from immunocompromised hosts were discussed. Acta Pathol Jpn 41: 668–672, 1991.     

Hepatic cytomegalovirus involvement in autopsy cases.

Hepatic involvement of cytomegalovirus (CMV) was studied in 15 autopsy cases with disseminated or solitary CMV infection. Formalin-fixed, paraffin-embedded tissue sections were examined by immunohistochemistry (IHC) using a monoclonal antibody against early and late CMV antigens, and by in situ hybridization (ISH) using biotinylated CMV DNA probes. Three cases showed cytomegalic cells in liver sections by conventional staining, five showed hybridization with CMV DNA probes and seven showed reaction with the monoclonal antibody in the liver. CMV infection was detected not only in cytomegalic cells but also in many non-cytomegalic cells by IHC and ISH, proving these techniques to be superior to routine histological examination. The inflammatory reaction in the liver was not prominent. The reason for the weak inflammatory response in the liver of our present cases, and the possible availability of IHC and ISH for analysis of liver biopsy and bronchoalveolar lavage specimens from immunocompromised hosts were discussed.     

Sensitivity and specificity of various morphological features of cervical condylomas. An in situ hybridization study

Fifty-seven cervical biopsy specimens or endocervical curettings showing condyloma, changes suggestive of condyloma, or no changes of condyloma were analyzed for presence of nuclear atypia (nuclear enlargement and irregularity in superficial epithelium), presence of multinucleated cells, and presence of perinuclear cytoplasmic clearing in superficial squamous epithelium. The findings were correlated with results of in situ hybridization with biotin-labeled human papillomavirus DNA probes. Moderate nuclear atypia was significantly more specific than perinuclear cytoplasmic clearing and 100% sensitive for predicting cases positive for human papillomavirus. Of the various morphological features analyzed, perinuclear cytoplasmic clearing had the lowest specificity for predicting positive results on in situ hybridization.     

Kaposi's sarcoma in acquired immune deficiency syndrome (AIDS)

Of 22 patients with Kaposi's sarcoma, 16 had the acquired immune deficiency syndrome (AIDS). The histological pattern in AIDS differs from the more familiar classical Kaposi's sarcoma. The features most useful in making the diagnosis are: dissection of collagen; lymphatic vessel like spaces; angiomatoid lesions; premonitory sign; and spindle cell proliferation. It is important to examine multiple levels of small biopsy specimens and to be cautious in making the diagnosis of patch Kaposi's sarcoma in the presence of recent or healed ulceration and at sites of previous trauma. Only four of 16 patients with AIDS had evidence of systemic Kaposi's sarcoma, supporting the view that Kaposi's sarcoma in AIDS does not necessarily have an aggressive clinical course.     

Frequency and anatomic distribution of lymphadenopathic Kaposi's sarcoma in the acquired immunodeficiency syndrome: an autopsy series

Histologic material from 52 autopsies of persons who had died of the acquired immunodeficiency syndrome (AIDS) were reviewed. The study group included 23 Haitians, 19 homosexual men, five intravenous drug abusers, two hemophiliacs (type A), and three persons at unknown risk. Nineteen of the patients (36.5 per cent) had typical Kaposi's sarcoma alone, but 49 (94.2 per cent) had the inflammatory variant of Kaposi's sarcoma as well as typical Kaposi's sarcoma. Inflammatory Kaposi's sarcoma was found in all risk groups studied. In all cases of typical Kaposi's sarcoma, histomorphologic transitions of inflammatory Kaposi's sarcoma to typical Kaposi's sarcoma were observed. Lymph nodes and spleen were the organs most commonly involved by both typical and inflammatory Kaposi's sarcoma. The findings indicate that Kaposi's sarcoma is more common and has a wider morphologic spectrum in AIDS than is generally appreciated.     

Chemiluminescent in situ hybridization for the detection of cytomegalovirus DNA.

A chemiluminescent in situ hybridization assay that could combine the sensitivity of chemiluminescent substrates, the specificity of digoxigenin-labeled probes, and the spatial morphological resolution and localization of the signal of the in situ hybridization was developed for the detection of cytomegalovirus (CMV) DNA. CMV DNA in cultured CMV-infected cells and in different clinical samples (tissue sections and cellular smears) was detected using digoxigenin-labeled probes constructed in our laboratory that were immunoenzymatically visualized employing anti-digoxigenin Fab fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal from the hybrid formation was detected, analyzed, and measured with a high performance, low light level imaging luminograph apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Increasing values of emitted photons per second per infected cell, corresponding to the presence of hybridized CMV DNA, could be found in infected cells fixed at various times after infection, following the CMV replication cycle. When the assay was performed on different clinical samples from patients with acute CMV infections, CMV DNA was detected in all positive samples tested, both in cellular samples and in frozen and paraffin-embedded tissue sections, proving specific and sensitive. The chemiluminescent in situ hybridization assay developed in this work can be a useful tool for a sensitive and specific diagnosis of viral infection and can be easily adapted to detect and study any specific gene sequence inside the cells. The assay may also be promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.     

Diagnosis of lung disease in acquired immune deficiency syndrome: biopsy or cytology and implications for management.

One hundred and twenty consecutive bronchoscopic examinations were carried out on 80 patients with acquired immune deficiency syndrome (AIDS) between January 1982 and December 1986. Ninety one paired biopsy and cytology specimens from 72 of these patients were analysed. There was no significant difference between biopsy and cytology in diagnosing Pneumocystis carinii pneumonia (0.95 greater than p greater than 0.1). In 10 cases P carinii pneumonia was diagnosed by biopsy but not cytology and in seven cases by cytology but not biopsy. Nineteen patients had multiple infections or Kaposi's sarcoma. Biopsy was more useful than cytology in the diagnosis of other infections (n = 20) and Kaposi's sarcoma (n = 2) with positive cytological correlation in only three of the infections. Biopsy and cytology together have a diagnostic yield of 78.3%. We conclude that all patients presenting with respiratory disease who have, or are in a high risk group for, AIDS should be examined by bronchoscopy at an early stage with both cytology and biopsy.     

Detection and localization of human papillomavirus DNA in human genital condylomas by in situ hybridization with biotinylated probes

We have examined the distribution of human papillomavirus (HPV) DNA in paraffin sections of humans warts by in situ hybridization with biotin-labeled DNA probes. Recombinant plasmid DNAs (HPV-1, -6, -11, -16) were labeled by nick translation with biotinylated deoxyuridine triphosphate. Paraffin sections were hybridized with the probes for 18 h in stringent or non-stringent conditions, and DNA-DNA hybrids were detected by immunocytochemistry. Paraffin sections of warts were also examined for the presence of HPV capsid antigen with the avidin-biotin peroxidase complex method for immunocytochemistry. HPV DNA was detected and localized in paraffin sections from a plantar wart, a laryngeal papilloma, and seven anogenital condylomas. The specific HPV type present in each lesion was determined by hybridization under stringent conditions with the homologous DNA probe. The papillomas were found to contain many more cells with replicating virus DNA, as demonstrated by in situ hybridization, than was apparent from the number of cells containing detectable virus antigen. In situ hybridization with biotin-labeled probes is an effective technique for the identification of HPV infection in routinely collected and processed tissue specimens.     

The histopathologic identification of CMV infected cells in biopsies of human renal allografts. An evaluation of 100 transplant biopsies by in situ hybridization

In order to determine the incidence and significance of CMV infected cells within human renal allograft biopsies 100 transplant biopsies were examined for the presence of CMV DNA within the renal tissue specimens using the in situ hybridization technique. In 41 cases CMV infected cells were predominantly found within proximal tubular epithelial cells, although typical nuclear inclusion ("owl eyes") were absent. In only one case was CMV detected within a few glomerular cells. The presence of CMV infected cells within allograft biopsies does not correlate with active CMV infection of the patients at the time of biopsy. There are no significant differences in the distribution of primary and secondary CMV infections between patients with positive and negative biopsy findings. No significant differences as to the histological alterations between CMV infected and non-infected biopsies could be found. The data give evidence that the renal allograft is more often affected by CMV than is generally appreciated. The in situ hybridization technique may be useful for the fast detection of latently CMV infected cells in renal transplants and thus may influence the choice of therapeutic steps early after transplantation. Furthermore, it may facilitate the diagnosis of interstitial nephritis due to virus infection if typical nuclear inclusions in routinely stained tissue sections are absent.