Detection of CMV in urine: comparison between DNA-DNA hybridization, virus isolation, and immunoelectron microscopy

Rapid detection of CMV-DNA in urine specimens by dot-blot hybridization was compared to conventional virus isolation and to virus identification using solid-phase immunoelectron microscopy (SPIEM). To detect viral DNA, 32P-labeled EcoR1 J fragment of CMV-DNA was used as a probe in the hybridization assay. In addition, DNA extracted from infected human embryo fibroblasts (amplified DNA) was also hybridized to the same probe. Urine specimens were obtained from 10 renal transplanted patients, seven premature infants, three family members, and five children suspected of CMV infection. CMV was isolated from 10 urine specimens and SPIEM detected viral particles in nine specimens. Ten positive samples were identified as such by hybridization with DNA extracted directly from urine specimens, while hybridization with amplified DNA yielded 17 positives. Only in one urine specimen, positive by virus isolation and SPIEM, DNA was not detected by the hybridization assays. Elevated IgG or IgM-specific antibodies were found in 10 patients. Hybridization with amplified DNA proved the most sensitive and relatively rapid assay, as compared with direct DNA detection in urine, tissue culture isolation, SPIEM, or serologic tests.     

A hybrido-immunocytochemical assay for the in situ detection of cytomegalovirus DNA using digoxigenin-labeled probes

A non-radioactive hybrido-immunocytochemical assay for the detection of cytomegalovirus (CMV) DNA in infected cells was developed. Two different DNA fragments belonging to the repeated sequences of CMV genome were used to construct the hybridization probe. The probe was constructed by incorporating deoxyuridine triphosphate labeled with digoxigenin. The in situ hybridized CMV DNA probe was immunocytochemically visualized by anti-digoxigenin. Fab fragments labeled with alkaline phosphatase. This procedure permitted the DNA detection, in the nuclei of infected cells fixed at 48 h after infection, of the Towne CMV reference strain and 21 different laboratory-isolated CMV strains. Our assay demonstrated a high specificity, sensitivity and reproducibility.     

ompA gene in the detection of Escherichia coli and other Enterobacteriaceae by nucleic acid sandwich hybridization.

The applicability of the DNA sandwich hybridization method to detection of bacterial DNA from crude samples is demonstrated using Escherichia coli as a model. In sandwich hybridization the sample DNA mediates the binding of a labeled probe DNA fragment to a second DNA bound on filter. For this study the DNA reagents were prepared by subclonings from a recombinant plasmid containing the E. coli K-12 ompA gene and an adjacent fragment of E. coli DNA. The 5' half of the ompA gene (738 base pairs) in pBR322 served as the filter reagent. The 3' half of the ompA gene (300 base pairs) and the adjacent 1,500 base pairs of E. coli DNA were cloned into the single-stranded phage vector M13mp7, and the resulting recombinant phage DNA was labeled with 125I and used as probe in the hybridizations. For maximal hybridization the DNA reagents had to be present in excess of the sample nucleic acid, which was preferably fragmented before testing. In the optimized test, 3 X 10(6) molecules of E. coli DNA from lysed cells were detected by an overnight reaction; the sensitivity of the test was not affected by the presence of 10(9) unrelated bacteria. With the ompA reagents, all members of the family Enterobacteriaceae tested were detected even if the sensitivity was decreased as compared with that for the homologous bacteria. With all other bacteria, including aerobic and anaerobic species of clinical importance, the test was negative. These findings suggest that it may be possible to find group-specific reagents to be used in diagnostic bacteriology.     

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.     

Nucleic acid sandwich hybridization in detection of HPV 16 DNA: technique and its clinical application

Nucleic acid sandwich hybridization technique was used for detection of HPV 16 specific DNA in cervical scrapes. Alternating HPV 16-PstI fragments were cloned into plasmid pBR322 and phage M13mp10. pBR322-clones were used as 32P-labelled probe reagents and the M13mp10 clones as catching reagents in the assay. The detection limit of the test proved to be 1-5 X 10(5) HPV 16 molecules per test. A weak cross reaction was seen with HPV 31 DNA but not with the other types tested, e.g. HPV 6, 11, 18 and 33. Cervical scrapings obtained from 163 consecutive patients included in a prospective follow up study were analyzed for HPV 16 DNA with the sandwich hybridization method, dot-blot hybridization being used as a reference method. Sandwich hybridization assay detected 25 positive cases out of 163 specimens (15.3%). Of these 6 and 3 additional specimens were positive in dot-blot hybridization assay. HPV 16 DNA was related to higher PAP grades, and HPV 16 appeared more frequently in HPV CIN than HPV NCIN lesions. None of the infections caused by HPV 16 regressed, and 24% progressed during the follow up.     

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.     

Comparison of different techniques for detection of cytomegalovirus infection following bone marrow transplantation

A total of 317 different clinical samples obtained from patients following bone marrow transplantation and 56 blood and urine samples from seronegative control persons were screened for the presence of human cytomegalovirus (CMV) using virus culture and slot-blot hybridization. Immunohistochemical techniques using monoclonal antibodies to various viral antigens and in situ hybridization techniques were also utilised for detection of CMV in tissue samples. In cell suspensions of blood, bone marrow and effusions, and in liver biopsies, CMV DNA could be demonstrated more often by slot-blot and in situ hybridization techniques than by virus culture or immunostaining of viral antigens. For detection of CMV in lung biopsies and other clinical samples containing mainly cell-free virus, such as urine, bronchial lavage and throat washings, virus culture was found to be at least as sensitive as the hybridization techniques. Immunostaining proved to be a fast and sensitive technique for detection of CMV in tissues and may thus provide additional information about viral replication and clinical relevance of the virus infection.     

Detection of Pseudomonas pseudomallei by PCR and hybridization.

A molecular method for the detection of Pseudomonas pseudomallei was developed on the basis of the differences in the 23S rRNA sequences of related species of the genus Pseudomonas. An 18-base oligonucleotide probe, designed following partial sequencing of 23s ribosomal DNA (rDNA), was used for the identification and detection of P. pseudomallei either by hybridization or by direct PCR. Optimal detection was obtained by hybridization of the probe with PCR-amplified rDNA rather than with total genomic DNA or colony blots. One nanogram of template DNA amplified in a PCR mixture containing 14% glycerol could be detected in slot blots hybridized with the digoxigenin-labelled probe and the lumigen PPD detection system. Amplified rDNA sequences from 41 P. pseudomallei strains of various origins hybridized with the probe. The probe also hybridized with three Pseudomonas mallei reference strains under conditions of high stringency but failed to hybridize with amplified rDNA sequences from other closely related Pseudomonas spp. PCR with a conserved primer and the 18-base oligonucleotide probe (direct PCR) specifically amplified P. pseudomallei and P. mallei. By using these methods, approximately 10(4) P. pseudomallei cells per ml could be detected in artificially inoculated blood samples and in blood dried on filter paper following Chelex extraction. The detection limit in blood was increased to 10(2) cells per ml by concentration of bacteria from 0.5 ml of blood or by a 24-h blood culture enrichment prior to PCR. Approximately 10(3) cells per ml were detected in seeded sputum samples. The detection times by direct PCR and indirect PCR and then probe hybridization were approximately 5 h and 24 h, respectively. These results indicate that amplification of conserved rDNA sequences by PCR directly or by hybridization with a probe to PCR fragments offers promise for the detection of P. pseudomallei and P. mallei.     

Usefulness of blood and urine samples collected on filter paper in detecting cytomegalovirus by the polymerase chain reaction technique

A rapid test for the diagnosis of congenital CMV infection is still needed. This study evaluated the usefulness of dried blood and urine samples collected on filter paper for detecting cytomegalovirus (CMV) by the polymerase chain reaction (PCR) assay compared with the use of liquid urine. Samples were obtained from 332 infants aged 1-7 days. Liquid urine samples were collected into bags, cultured in human fibroblasts, and processed using a multiplex PCR technique. Dried urine samples were obtained by placing a piece of filter paper in contact with the infant's genitals. The heels of neonates were punctured and capillary blood was blotted onto filter paper and dried. Dried blood and urine specimens were analyzed by multiplex PCR and nested-PCR assays. A diagnosis of congenital CMV infection was established by isolating the virus, and by detecting viral DNA in the liquid urine. Of the 332 liquid urine samples collected from 332 neonates, seven (2.1%) were positive for CMV and 325 were negative, by both cell culture and PCR assay. In dried samples, CMV DNA was detectable only with a nested PCR assay. Compared with known CMV infection status, 5/7 (71.4%) neonates were positive for congenital CMV infection using dried blood samples. All 325 uninfected neonates were negative. In the dried urine samples, 4/4 CMV-infected infants gave positive tests, and all 262 uninfected infants were negative. Although further improvements in sample collection and/or processing are still needed, PCR testing on dried urine or blood collected on filter paper is a promising approach in the diagnosis of neonatal CMV infection.     

Rapid detection of cytomegalovirus by fluorescent monoclonal antibody staining and in situ DNA hybridization in a dram vial cell culture system.

By using dram vial cell culture methods, three commercially available tests for cytomegalovirus (CMV) detection were compared: direct fluorescent monoclonal antibody staining for CMV-specific early and late antigens (direct FA), indirect fluorescent monoclonal antibody staining for a CMV-specific early antigen (indirect FA), and in situ DNA hybridization with a biotinylated CMV-specific DNA probe kit (DNA probe). Of those tests, only the indirect FA provided consistent, reliable virus detection within the initial 24 h postinfection for serial 10-fold dilutions of CMV AD169 (laboratory strain) and for three selected urine samples. However, when used prospectively, the indirect FA failed to detect virus within the initial 10 days postinfection in 15 of 78 consecutive specimens that were eventually positive by cell culture. Although the indirect FA was more sensitive than the direct FA or DNA probe, its utility appeared limited to specimens with high CMV concentrations. On the basis of these data, we recommend that indirect FA be reserved as an adjunct to standard cell culture for selected samples in diagnostic hospital laboratories.     

DNA of 21 clinical cytomegalovirus strains detected by hybridization to cloned DNA fragments of laboratory strain AD-169

Nine distinct DNA probes prepared from cloned DNA fragments of the cytomegalovirus (CMV) strain AD-169, each representing a relatively small portion of the total genome, were able to detect all 21 epidemiologically distinct clinical isolates of CMV that had been passaged in tissue culture. All probes were of comparable sensitivity in the detection of CMV, and under the hybridization conditions used, no probe gave an unusually high background with uninfected host cells.     

Quantitation of cytomegalovirus DNA by blot hybridization in blood leukocytes of viremic patients

We describe a technique for quantitation of viral DNA in blood leukocytes during viremic infection with human cytomegalovirus (CMV). Using a cloned subgenomic DNA probe and a blot hybridization assay, small amounts of viral DNA within samples of leukocyte DNA could be quantitated reproducibly using a videodensitometer. Critical components of the assay were: (1) a direct relationship between optical density and known amounts of viral DNA diluted in cellular DNA as positive standard samples and, (2) determination of the proper duration of autoradiographic exposure. The technique was sufficiently sensitive to detect 10 picograms of CMV DNA in the presence of microgram quantities of host cell DNA. In addition, samples containing minute amounts of CMV DNA could reliably be distinguished from samples that were negative. We have used the technique to characterize the pathogenesis of CMV viremia and to monitor the effects of antiviral chemotherapy. This procedure could easily be applied to pathogenetic studies of a variety of other infectious agents.     

Detection of bacteria by hybridization of rRNA with DNA-latex and immunodetection of hybrids.

A novel nucleic acid hybridization assay with a DNA probe immobilized on 1.25-micron-diameter latex particles was developed. Hybridization of the immobilized probe DNA with sample rRNA was complete in 10 to 15 min. Alkaline phosphatase-labeled anti-DNA-RNA was allowed to bind to the DNA-RNA hybrids on the latex particles. Then the latex was collected on a small glass fiber filter pad, and bound alkaline phosphatase was quantitated by reflectance rate measurement. The method detected a broad range of bacterial species and had a detection limit of 500 cells per assay. The assay was used to screen urine samples for bacteriuria and had a sensitivity of 96.2% compared with conventional culture at a decision level of greater than or equal to 10(4) CFU/ml. The hybridization method could have broad application to the detection of bacteria and viruses.     

Demonstration of cytomegalovirus nucleic acids in the coronary arteries of transplanted hearts.

Cytomegalovirus (CMV) infection has been associated with the development of accelerated arteriosclerosis in heart transplant recipients. This association prompted the authors to examine, using in situ hybridization techniques, the coronary arteries of 19 patients who had received heart transplants for the presence of CMV nucleic acids. Two probes were employed on formalin-fixed paraffin-embedded tissues. The first was an S35-labeled nick translated probe derived from CMV DNA. CMV nucleic acids were identified in the arterial intima of 5 (45%) of 11 transplanted hearts examined with this probe. Hybridization to cells morphologically consistent with endothelial cells, lymphocytes, and smooth muscle cells was identified. Hybridization was not detected in any of the hearts when a control probe, a vector without CMV DNA, was employed. The second probe that was used was a tritium-labeled strand-specific ribo-probe derived from the immediate early (IE1) gene of CMV. Using this second probe, hybridization was detected in the coronary arteries of three of five hearts that were positive with the DNA probe, in one of the hearts that was negative with the DNA probe, and in three (38%) of eight additional transplanted hearts. The pattern of hybridization was the same as that seen with the DNA probe, and no hybridization was detected when a control sense probe for the IE1 gene of CMV was employed. These results suggest that CMV nucleic acids are present in the coronary arteries of heart transplant recipients.     

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.     

The presence of cytomegalovirus nucleic acids in arterial walls of atherosclerotic and nonatherosclerotic patients.

The presence of cytomegalovirus (CMV) nucleic acids was demonstrated in abdominal aortas and femoral arteries of patients with and without atherosclerosis by dot blot and in situ DNA hybridization using a DNA probe derived from immediate early genomic regions. Viral antigens could not be detected by immunohistochemistry and infectious virus could not be recovered from the arterial wall by virus isolation techniques. The high percentage (55%) of vascular wall specimens containing CMV nucleic acids, in atherosclerotic as well as in control material and the location of CMV-containing cells in arteries without gross changes indicative of atherosclerosis suggest that the human arterial wall may be a site of latency for this virus.     

Sensitivity of in situ hybridization techniques using biotin- and 35S-labeled human papillomavirus (HPV) DNA probes

In order to evaluate the sensitivity of our modified in situ DNA hybridization technique using biotinylated probes, formalin fixed, paraffin embedded biopsies from 20 cervical lesions known to contain human papillomavirus (HPV) DNA were re-examined by the technique using both 35S-labeled- and biotinylated HPV DNA probes. The probe concentrations as well as the detection limits of biotin probing were screened by spotting known amounts of HPV 16 DNA on nylon filter, and allowed to hybridize with biotinylated HPV 16 DNA probe. By this method, 4 pg of HPV 16 DNA could be detected using a probe concentration of 0.2 micrograms/ml. HPV DNA could be demonstrated in all 20 biopsies with both hybridization techniques. However, signals in subrabasal cells were detected more frequently with biotin- than with 35S-labeled probes. Additional experiments were performed using three cervical cancer cell lines (with known copy numbers of HPV DNA), to assess the detection limits of HPV infections by the in situ hybridization techniques. The CaSki cells (500-600 HPV 16 copies/cell) were unequivocally positive with both labelling systems. HeLa cells (10-50 HPV 18 copies/cell) were positive with the biotin probing in 10/10 smears, as compared to 7/10 smears when 35S-labeled probes were used. Radioactive probing was inferior to biotinylated probing in detecting the signals in SiHa cells (1-2 HPV 16 copies/cell). This is because even weak background signals could mask true positive signals when 35S-labeled probes are used. In contrast, no background is generated with the biotinylated probes, detected with streptavidin-biotinylated alkaline phosphatase complex. In situ hybridization with biotinylated DNA probes is as sensitive as techniques using 35S-labeled probes for detecting HPV infections in routine cervical biopsies or smears.     

High prevalence of latently present cytomegalovirus in arterial walls of patients suffering from grade III atherosclerosis.

The presence of cytomegalovirus (CMV) nucleic acids was demonstrated in arterial walls of patients with grade III and with maximally grade I atherosclerosis by dot blot and in situ DNA hybridization and by polymerase chain reaction (PCR) using probes and primers derived from immediate early (IE) and late (L) genomic regions. The presence of the complete viral genome could be demonstrated by both dot blot DNA hybridization and PCR. IE mRNA but not L mRNA could be demonstrated by in situ DNA hybridization, indicating the presence of latent CMV in the human arterial wall. By PCR 90% of the samples obtained from atherosclerotic patients were shown to contain viral nucleic acids as compared to 53% of patients with maximally grade I atherosclerosis, thus substantiating a role for this virus in the pathogenesis of atherosclerosis.     

Cytomegalovirus detection by nonisotopic in situ DNA hybridization and viral antigen immunostaining using a two-color technique

Rapid methods of specific viral diagnosis in formalin fixed, paraffin embedded tissues include identification of viral incusions in routinely stained histologic sections, immunologic staining of viral antigens, and in situ nucleic acid hybridization. To correlate in situ hybridization with immunologic detection methods, sequential two-color staining was used on tissues from 12 patients, each containing characteristic cytomegalovirus (CMV) inclusions, using a biotinylated CMV DNA probe in an avidin-alkaline phosphatase-linked reaction followed by avidin-biotin complex immunoperoxidase staining of CMV antigen. CMV genetic material was seen in all 17 tissues. CMV antigen was detected in 11 of 17 tissues (65%). The DNA hybridization technique provided more intense staining, detected greater numbers of inclusions, and had less background staining than the immunoperoxidase technique. The alkaline phosphatase reaction product was stable through subsequent immunostaining steps, and immunologic reactivity of CMV antigen was not significantly reduced by prior hybridization steps. CMV DNA probe was localized predominantly within cell nuclei, while CMV antigen immunostaining was predominantly cytoplasmic. It was concluded that sequential in situ hybridization and immunocytochemistry can be performed on standard histologic sections. Furthermore, it is likely that the majority of CMV nucleic acid detected by this tissue hybridization technique is unencapsidated, intranuclear viral DNA and not DNA contained within complete CMV nucleocapsids.     

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.