Detection of human papilloma virus (HPV) DNA in genital biopsy specimens by in situ hybridization with digoxigenin-labeled probes.

The presence of human papillomavirus (HPV) nucleotide sequences in paraffin sections of genital biopsies was examined by in situ hybridization using non-isotopic, digoxigenin-labeled probes representing HPV types 11, 16 and 18. Digoxigenin-labeling of the probes was performed using DNA labeling and a commercially provided detection kit. Hybridization was performed under stringent conditions. The hybrids were detected by using anti-digoxigenin alkaline phosphatase conjugate and visualized with enzyme catalyzed color reaction. In situ hybridization with digoxigenin-labeled probes was a useful technique for identification of HPV infection. The results were compared with the results obtained with radiolabeled DNA probes. The sensitivity of the digoxigenin-labeled probes was equal to the sensitivity of the radiolabeled probes. The background with digoxigenin-labeled probes was very low. Using nonradioactive probes the localization of hybrids at the cellular level was better than 35S-labeled probes.

Solid-phase enzyme-immunoassay for the detection of herpes simplex virus antigens in clinical specimens.

An indirect solid-phase enzyme-immunoassay (EIA) for the detection of herpes simplex virus (HSV) antigens in clinical specimens was developed. Rabbits and guinea pigs were hyperimmunized with highly purified nucleocapsids of HSV type 1. Microtitre plates were coated with 0.25 microgram of guinea pig anti-herpes simplex type 1 immunoglobulins per well. Clinical specimens, diluted in phosphate buffered saline containing fetal calf serum and detergents, were sonicated and incubated in the test wells overnight at 37 degrees C. Rabbit anti-HSV immunoglobulins were added as a secondary antibody at a concentration of 3.2 micrograms per well, and peroxidase conjugated swine antibodies against rabbit immunoglobulins, diluted 1:1,000, were used as a fourth layer. Clinical specimens which were sent for virus isolation or for isolation of Chlamydia trachomatis were tested by the developed assay and 20 out of 27 isolation positive specimens were found positive by EIA. Five out of 67 specimens negative by isolation gave positive results by EIA. The specificity of the results was confirmed by a control test using wells coated with normal guinea pig immunoglobulins. The test detected antigens from both serotypes of HSV. Cross reactions with varicella-zoster- or with cytomegalovirus were not found, and antigens from uninfected cells did not result in false positive results.

Typing of herpes simplex virus isolates with monoclonal antibodies and by nucleic acid spot hybridization.

Fifty-one clinical isolates of herpes simplex virus (HSV) were typed by an enzyme immunoassay (EIA) using mouse monoclonal antibodies, by DNA spot hybridization, and by restriction enzyme analysis using restriction endonuclease Eco RI. Extracts of VERO cells infected with the isolates were used for coating microtitre plates or denatured and spotted onto nitrocellulose filters. Viral antigens passively adsorbed to microtitre plates were detected by an indirect EIA using mouse monoclonal antibodies specific for HSV type 1 (HSV-1) or HSV type 2 (HSV-2). Spotted DNA was hybridized with 32P-labeled probes containing Hind III/Sal I-fragments of either HSV-1 or HSV-2 DNA and bound radioactivity was detected by autoradiography and counted in a liquid scintillation counter. All the three methods gave identical results for the 51 isolates studied. Twenty-six isolates were identified as HSV-1 and 25 as HSV-2. An additional 30 specimens were tested only by EIA and hybridization. Results by both techniques were in complete agreement.

Clinical scoring system in the evaluation of adult pharyngitis.

OBJECTIVE: To compare results of a clinical scoring system for diagnosis of group A streptococcal pharyngitis with microbiologic results, when several different pharyngeal pathogens were tested simultaneously. DESIGN: Evaluation of clinical manifestations of 106 adult patients with pharyngitis of different microbial origin. SETTING: General private practice; Health Center Pulssi, Turku, Finland. PATIENTS: Adult patients whose chief complaints were sore throats. MAIN OUTCOME MEASURE: A symptom score that was assigned to each patient according to the total number of certain signs and symptoms that are postulated to increase the probability of group A streptococcal pharyngitis and blood measurements for infection. RESULTS: The highest symptom scores, 3 and 4, were found in 21 patients. These patients had pharyngitis due to group A streptococcus (four patients), group C streptococcus (four patients), group G streptococcus (two patients), group F streptococcus, Mycoplasma pneumoniae, Chlamydia pneumoniae, influenza A virus, influenza B virus, herpes simplex type 1 virus (two patients), and coxsackie B4 virus. No pathogen could be identified from three of the 21 patients. The C-reactive protein values and the leukocyte counts were raised significantly more often in streptococcal infections than in infections of other origin; the P values were .00016 and .028, respectively. CONCLUSION: Use of a clinical scoring system alone for diagnosis of pharyngitis may lead to improper use of anti-microbial agents. There is a need for accurate microbiologic diagnostic procedures in general practice to determine proper treatment of pharyngitis as well as to test the effect of antibacterial and, in the future, antiviral treatment in respiratory tract infections.

Human papillomavirus detection from the female genital tract: combined vs. separate scrape methods.

Because genital human papillomavirus (HPV) infections tend to be multifocal, it was studied how effective one combined specimen is in detecting HPV-DNA from the lower female genital tract. The study population consisted of 50 patients referred to a colposcopy clinic for a suspected condylomatous and/or dysplastic lesion. From half of the patients, a separate scrape from the cervix, vagina and vulva was taken first followed by a combined scrape representing all the genital sites, and from the other half, vice versa. HPV-DNA (types 6, 11, 16 and 18) was identified using the AffiProbe hybridisation test. Thirty-six specimens collected from 17 patients were positive for HPV-DNA. A multifocal infection was demonstrated in at least 11/17 (65%) HPV-DNA positive patients. The combined scrape was the most informative specimen, revealing 75% of all HPV-DNA-positive patients. It was concluded that HPV-DNA can reliably be detected from the female genital tract in a simple way from one combined specimen.

Surveillance of human immunodeficiency virus (HIV) antibodies in medicolegal autopsies in Finland--monitoring early changes in HIV-seropositivity among risk groups and average population.

In order to cooperate with voluntary screening programs aimed at the surveillance of the HIV epidemic in Finland, we have studied medicolegal autopsies for HIV antibodies since 1986 using an enzyme immunoassay on postmortem sera. The investigation covered 47.4% and 39.2%, respectively, of all deaths under the age of 65 years in the metropolitan areas of Helsinki and Turku--two cities on the densely populated southern coast of Finland from which most HIV infections have thus far been detected. Nine HIV-positive cases (0.12%) were detected among the 7305 medicolegal autopsies tested in 1986 to 1990. This figure is higher than the prevalence of 0.01 to 0.03% in voluntary screening programs for the general population would suggest. Seven of our cases had previously tested positive, and two were previously unknown cases, indicating that people at high risk are clustered in the medicolegal autopsy series. Of the six cases in an early stage of infection, three committed suicide suggesting the importance of HIV-screening in suicide cases in tracing symptomless HIV carriers. Five of the cases were detected in 1990, a year when the number of new HIV infections had more than doubled compared to the previous two years. This suggests that testing of medicolegal autopsies as surrogate tests for the population gives useful information even in low-prevalence areas like Finland. Such testing has none of the ethical problems of many other back-up surveys, and may be particularly sensitive to early changes in epidemiology.

Quantitative studies on adsorption, elution, and haemagglutination of vesicular stomatitis virus.

Radioactively-labelled VSV, both Indiana and New Jersey serotypes, were rapidly and efficiently adsorbed onto goose erythrocytes, but only within a narrow range of pH and at low temperatures. Under optimal conditions for adsorption, both infectivity and haemagglutinating activity of VSV were reduced. A basic difference between the two serotypes was identified: adsorption and haemagglutination of the Indiana serotype were stable even at pH 9.0 and at 37 degrees C, whereas New Jersey serotype was eluted slowly at +0 degrees C and at pH 6.4 and rapidly at a higher temperature and pH. The rapid elution offers a useful method for purification of New Jersey serotype. Particle counting of VSV showed that 10 to 50 virus particles were required for one PFU. Haemagglutination was not a sensitive method for assaying small amounts of VSV; 3--20X10(7) virus particles or more than 10(7) PFU corresponded to one HAU.

Polyethylene glycol increases specificity of hybridization typing of HPV specimens.

Different hybridization conditions for the typing of human papillomaviruses (HPV) in clinical biopsy specimens were tested. The stringency of the hybridization reaction was varied by altering the formamide concentration in the solution. Two polymers, dextran sulphate and polyethylene glycol (PEG), were compared as accelerators of the hybridization reaction. The PEG-containing hybridization solution was found to be suitable for typing clinical HPV specimens. Single-stranded RNA probes proved to be more specific than DNA probes in typing clinical specimens. Specimens that were positive both for HPV6 and HPV11 in spot hybridization were confirmed by Southern hybridization. In total, 467 biopsy specimens from genital, anal, oral, aural and nasal lesions were examined for HPV6, HPV11, HPV16 and HPV18 DNA by spot hybridization. Approximately one-third of the specimens were positive for these types.

Characterization of a hamster brain cell line persistently infected with measles virus.

A persistent infection of measles virus in hamster brain cell cultures was established. Hamster brain cells were transformed with a human papovavirus strain BK and infected with a wild-type measles virus in order to get the cell line persistently infected with measles virus. About 75 per cent of these M-HB/MVB-cells were measles virus-infected. The cells released only small amount of infectious virus and produced low levels of interferon-like activity into the growth medium. During the first 50 passages no large syncytia typical of a lytic measles virus infection were seen. The products of measles virus infection in the cells and in cell culture fluids were followed at two temperatures. Another cell line persistently infected with measles virus (Lu-carrier-cells, 28) was investigated in parallel. In both cell lines measles antigens were cytoplasmic, but during the observation period large amounts of measles nucleocapsid accumulated in the nuclei of the M-HB/MVB-cells. The virus-specific protein synthesis in M-HB/MVB-cells was weak and the intracellular amount of immunoreactive measles nucleocapsid was only 50 per cent (600 ng/10(5) infected cells) of the (1200 ng/10(5) cells) found in Lu-carrier-cells. Also the release of nucleocapsid was minimal in hamster brain cells. The decreased temperature had no clear-cut effect on virus-specific protein synthesis or on the release of the virus-specific products.

Herpes simplex virus detection by macroscopic reading after overnight incubation and immunoperoxidase staining.

Human diploid foreskin fibroblast cells grown in 24-well plates were inoculated with clinical specimens by centrifugation at 1,000 X g for 45 min. Cultures were incubated at 37 degrees C overnight, fixed, and stained with peroxidase-labeled monoclonal antibodies against herpes simplex virus types 1 and 2. Stained plaques of infected cells were large enough to be detected with the naked eye, and microscopic examination did not reveal any further positive specimens. The method was compared with standard isolation in human fibroblasts grown in shell vials and inoculated by centrifugation at 4,000 X g, observed microscopically for the occurrence of typical cytopathogenic effect three times a week for 10 days, and then typed by enzyme immunoassay. Of the 289 specimens tested, 105 were positive and 174 were negative by both methods. Six specimens were positive by standard isolation only, two of them containing varicella-zoster virus, and two specimens were stored frozen before being tested by immunoperoxidase staining. Two specimens found negative by standard isolation were positive by immunoperoxidase staining. For two specimens negative by immunoperoxidase staining, the standard isolation cultures were lost due to microbial contamination. Forty-two specimens found positive by standard isolation were clearly positive when stained only 8 h after inoculation. By standard isolation, positive results were reported on the average 3 to 4 days after inoculation, whereas by immunoperoxidase staining the result was available within less than 24 h. Immunoperoxidase staining of infected cells is a sensitive method for rapid laboratory diagnosis of herpes simplex virus infections, and 24-well plates are convenient for the handling of a large number of specimens.

Expression of the L2 and E7 genes of the human papillomavirus type 16 in female genital dysplasias.

The expression of the E7 and L2 genes of HPV 16 was studied in benign and precancerous female genital lesions to evaluate their role in the development of dysplasias. Ninety biopsy specimens from 70 patients, selected on basis of dot blot DNA hybridization, were included in immunohistochemical and in situ hybridization analyses. In the HPV 16 DNA positive cases, L2 mRNA and E7 mRNA were detected in biopsies from 24 and 21 patients, respectively. L2 mRNA was found in eight of 16 cases of condyloma and mild dysplasia, and in 13 of 14 cases of moderate to severe dysplasia. The figures for E7 mRNA were 6/16 and 13/14, respectively. We found L2 mRNA in four of 12 normal or condylomatous specimens and E7 mRNA in only one of these. The detection rates for L2 and E7 mRNAs increased along with the severity of the lesions (P = 0.0064 and P = 0.0001, respectively). The L2 protein was found in one condyloma and in 12 dysplasias, eight of which were moderate or severe. The L2-antibody-reactive cells were localized in superficial layers of the epithelium. The detection rate for L2 mRNA and especially for E7 mRNA increased along with the histopathologic grade of the lesion.

Comparison of four-layer radioimmunoassay and electron microscopy for detection of human rotavirus.

A four-layer radioimmunoassay (RIA) using polystyrene beads as the solid phase, anti-rota guinea pig IgG as primary antibody, anti-rota rabbit IgG as secondary antibody, and 125I-labelled sheep anti-rabbit immunoglobulin as indicator antibody has been developed for the detection of human rotavirus in stool specimens. A comparison was made of the developed RIA, routine electron microscopy, and research electron microscopy of 147 unconcentrated stool specimens from patients with infantile gastroenteritis. In routine electron microscopy 17 (11.6%) false-positive or false-negative results were obtained when compared with research electron microscopy. Each specimen positive in research electron microscopy was positive in RIA, and six additional RIA positives were found from 58 electron microscopy negative specimens. A confirmatory test was necessary to find out marginally positive but nonspecific reactions in RIA. The developed radioimmunoassay is slightly more sensitive than research electron microscopy of unconcentrated stool specimens and considerably more sensitive and more specific than routine electron microscopy.

Hemagglutination by BK virus, a tentative new member of the papovavirus group.

Some characteristics of hemagglutination (HA) by the BK virus, a new candidate for the papovavirus group, have been studied. Hemagglutinin prepared from cell cultures was found to be partially masked by inhibitors which could be dissociated from the virus by incubation at 37 C or by fluorocarbon extraction. Optimal conditions for HA are outlined. In routine tests, 0.5% human erythrocytes were used. The reaction was carried out at pH 7.0 on ice-water slurry. BK hemagglutinin receptors on human erythrocytes were found to be more resistant to neuraminidase than polyoma receptors. By gradient centrifugation analysis, two types of particles were found to be responsible for HA: (i) full, deoxyribonucleic acid-containing particles with a density of 1.325 g/cm(3) and (ii) empty capsids with a density 1.29 g/cm(3). Based on particle counting, one HA unit was calculated to correspond to 3 x 10(6) virus particles.

Central nervous system involvement in patients with rotavirus gastroenteritis.

Two children with rotavirus gastroenteritis are presented. The first case developed a fatal Reye's syndrome and the other one encephalitis with slow recovery. The rotavirus diagnosis was made in both cases by electron microscopy and a significant rise in antibody titres to Nebraska calf diarrhea virus was seen in one of the two patients.

Comparison of polyclonal and monoclonal antibodies to Actinomyces and Arachnia species.

Polyclonal (PoAbs) and monoclonal (MoAbs) antibodies were produced to Actinomyces israelii serotypes 1 and 2, to Actinomyces naeslundii, and to Arachnia propionica, and their specificities were studied by an enzyme immunoassay (EIA). All PoAbs except those to A. propionica reacted also with at least one other Actinomyces species. Only the MoAb to A. naeslundii proved to be more specific than the corresponding PoAbs. This MoAb did not crossreact with other Actinomyces or Arachnia species, nor with any other anaerobic or aerobic bacteria studied by inhibition EIA. Immunoblotting studies indicated that the antibody specific to A. naeslundii is directed against a large molecular weight antigen (greater than 150 kd), probably polysaccharide in nature. The produced PoAbs and MoAbs can be used for further analyses of the antigenic determinants of different Actinomyces and Arachnia species.

Comparison of smear specimens with biopsy specimens in a nucleic acid hybridization test for human papilloma virus (HPV) infection.

A total of 323 pairs of specimens from women with Papanicolaou class II or III cytology were examined for human papillomavirus (HPV) types 6, 11, 16 and 18 by spot hybridization. Each pair consisted of a representative biopsy specimen and a smear specimen from cervical, vaginal or, more rarely, vulvar lesions. We found a close correlation between HPV findings in biopsies and smears. In 83.9% (271) of the cases, both specimens were either positive for a given HPV type or negative. No discordant HPV-types in the two types of specimens were found. In 15.8% (51) of the cases, one specimen proved positive for a given HPV-type while the other specimen from the same patient was negative. In 8.0% (26) of the cases, the biopsy specimen proved positive and the corresponding smear specimen was negative. On the other hand, in 7.7% (25) of the cases we were able to detect HPV DNA in the smear specimen, whereas the corresponding biopsy specimen was negative. We suggest that a smear specimen would be advantageous for screening large groups of patients for the presence of a HPV infection in the genital tract. By using smear specimens together with biopsy specimens it is possible to maximize the number of HPV infection diagnoses.

Characterization of the common immunodominant antigenic determinant in the lipopolysaccharide of Bacteroides fragilis by a monoclonal antibody.

The target determinant of a monoclonal antibody (MoAb) to Bacteroides fragilis lipopolysaccharide (LPS) was characterized by inhibition enzyme immunoassay (EIA), immunoblotting (IB), immunofluorescence technique (IF) and electron immunocytochemical (EIC) technique. The MoAb has been shown to react positively with 96% of B. fragilis isolates. LPS preparations from 14 different B. fragilis strains were tested by EIA and IB. Two LPS preparations did not react in any of the tests. In both preparations the D-galactose was either lacking or present in low amount compared with the other LPSs. In addition, inhibition experiments with synthetic disaccharides confirmed that the target determinant is composed of beta-1,6-linked galactose disaccharide. EIC showed that the target of the LPS-MoAb is located on the surface of the outer membrane. These results show that the galactose chain present in LPS isolated from most B. fragilis strains contains the immunodominant antigenic determinant.

Four-layer radioimmunoassay for detection of adenovirus in stool.

A four-layer antispecies radioimmunoassay (RIA) was developed for the detection of adenovirus in stool specimens. Polystyrene beads were used as the solid phase, anti-adenovirus guinea pig immunoglobulin (1 microgram per bead) was used as the primary antibody, anti-adenovirus rabbit immunoglobulin (16 micrograms/ml) was used as the secondary antibody, and 125I-labeled sheep anti-rabbit immunoglobulin was used as the indicator antibody. A highly purified, crystallized adenovirus type 2 hexon antigen was used as the immunizing antigen for the production of hyperimmune sera. The sensitivity of the test was 1 ng of hexon protein per ml. Each of the 13 stool specimens positive for adenovirus by electron microscopy was positive for adenovirus by the RIA. Of 200 nonconcentrated stool specimens negative by electron microscopy, 14 additional specimens were positive by the RIA, increasing the detection rate from 6% by electron microscopy to 13% by the RIA. A confirmatory test was done on the RIA-positive, electron microscopy-negative specimens, and the test indicated a true specific result with each specimen. A confirmatory test was also done on each specimen with a low positive counts per minute value. The specificity of the RIA was further demonstrated by the fact that a positive result was found with only 3 of 295 specimens positive by the rotavirus RIA. In two of these three specimens, adenovirus and rotavirus were also detected simultaneously by electron microscopy, and the third specimen was from a patient with serological evidence for a dual infection. The adenovirus and rotavirus RIAs are now in a routine diagnostic laboratory, and in the 307 stool specimens tested during the first 5 months, the positive rate was 32% for rotavirus and 9.5% for adenovirus.

Involvement of the central nervous system in acute, uncomplicated measles virus infection.

A prospective study on acute, uncomplicated measles infection was carried out in 59 patients hospitalized for an ordinary measles infection. A clinical and serological diagnosis of an acute measles infection was made in all cases. Serial serum and cerebrospinal fluid (CSF) specimens were taken two to five times from each patient and tested for pleocytosis, albumin, and measles-specific antibodies. Pleocytosis was found in 18 patients (30%), usually shortly after the onset of rash. Nine patients had antibodies against measles virus in their CSF. Six of them seemed to have damage to the blood-brain barrier, but in two cases there was a very high serum antibody titer with a normal serum/CSF ratio. One patient had a local antibody production against measles virus in the central nervous system. Conventional electroencephalography (EEG) was recorded on 22 children, and a separate, quantitative EEG study with two to six consecutive recordings was also performed on a group of nine patients. Moderate or strong slowing of background EEG activity was found in 50% of the patients. In the consecutive recordings, the changes culminated a few days after the onset of rash. No correlation seemed to exist between the changes in the CSF and the age of the patient, on the one hand, and slowing of the EEG, on the other.