CMV PCR monitoring in leucocytes of transplant patients.

BACKGROUND: The presumed latency of cytomegalovirus (CMV) in leucocytes and the sensitivity of the polymerase chain reaction (PCR) raise a question of its clinical value. OBJECTIVES: To develop and standardize a CMV PCR as a diagnostic tool for CMV infection in solid organ and bone marrow transplant patients by comparing it to a likewise standardized isolation, rapid isolation and to clinical symptoms. STUDY DESIGN: The material comprised 822 EDTA peripheral blood samples from 96 solid organ and 119 bone marrow transplant patients. One sample from each of 21 healthy bone marrow donors and 25 blood donors were used as controls. Two million leucocytes were lysed and one-tenth of a volume was used in a nested PCR employing immediate early gene primers. RESULTS: The limit of detection was approximately 10 gene copies of a CMV DNA clone and 1 TCID(50) of extracted DNA from a cell suspension. The specificity was >/=0.99 when tested in CMV seronegative individuals. The positive and negative predictive values were 0.62 and 1.00, respectively. When PCR was compared to virus isolation/rapid culture in individual patients, PCR was positive more frequently in solid organ transplant patients than was CMV isolation/rapid culture, but the difference was not significant in bone marrow transplant patients. In isolation-positive patients, PCR became positive in samples taken 1-2 weeks earlier. In 54 solid organ transplant patients with PCR-positive samples, CMV-associated symptoms were present in 29/31 patients with CMV isolated from blood but in only 5/23 patients without viraemia. In 17 bone marrow transplant patients treated with ganciclovir, PCR became negative during or immediately after treatment in 14/20 (70%) episodes. This was true of 5/12 (42%) solid organ transplant patients. CONCLUSION: Screening of transplant patients with CMV PCR can be standardized at a clinically relevant level so that antiviral therapy can be instituted early.

Detection of CMV-matrix pp65 antigen in leucocytes by immunofluorescence as a marker of CMV disease.

The finding that active cytomegalovirus (CMV) infection is associated with an increased mortality after organ transplantation and the introduction of successful antiviral treatment render more urgent the development of rapid and accurate diagnostic methods. Rapid immunodiagnosis of active CMV infection was investigated by means of immunofluorescence staining of leucocytes by monoclonal antibodies. Monoclonals directed against the matrix protein, pp65, and ClonabR monoclonal antibody, which has been claimed to be directed against immediate early antigens, were used. All monoclonal antibodies directed against the matrix protein reacted equally well in staining of polymorphonuclear cells. Monoclonals described by Gerna et al. [1991] and by Pereira et al. [1982] also reacted more clearly with mononuclear cells. One hundred fifty heparinized blood samples were collected monthly from 82 patients within 3 months after transplantation. In addition, 132 EDTA blood samples were tested in connection with suspected CMV infection. The latter approach gave a better agreement between antigen detection and virus isolation. There was a relationship between active CMV infection and the finding of antigen-positive leucocytes (P = 0.002, Fisher's exact test) found within 1 month of one another. When the number of antigen-positive cells was taken into account, a relationship to severe CMV disease was detected (P < 0.001, Fisher's exact test). The antigen test was positive at an early stage during the development of severe CMV disease. This rapid method is useful for following the disease process of CMV and in determining when to initiate antiviral treatment.

[Study of the multiplication in cell cultures of two strains of para-influenza virus type 3. III. Multiplication in BHK 21 cells]. / Etude de la multiplication sur cultures de cellules de deux souches de virus paragrippal du type 3. Note III. Multiplication sur cellules BHK 21.

Study was conducted on the multiplication of two strains (C243 and D) of parainfluenza virus type 3 in BHK 21 cells. Multiplication curve of the virus was established and immunohistochemical aspects of the process were investigated. Chronological study of successive steps of the formation and development of viral components allowed to see that the virus multiplication rate is low in this cell system. The parainfluenza antigen became detectable by immunofluorescence in the infected cell perinuclear region after a relatively long eclipse period (18 h) and synthetized virus has few RNA and induced no inclusion information in the cytoplasm or the nucleus. However, an important nuclear participation was noted: 72 h after inoculation, nuclear fluorescence was observed, as well as a nuclear DNA rising and frequent aberrant mitoses. Comparison between the two investigated strains led to the observation that the autochthonous D strain induced more frequent aberrant mitoses and more important cell destruction than the C243 one. Differences were also noted as regards the infecting and hemagglutinating titers.

[Effect of immunization with an inactivated influenza vaccine, NIVGRIP, on the viral population and clinical course of patients with chronic non-bacterial pharyngitis]. / Effets de l'immunisation à l'aide du vaccin inactivé contre la grippe NIVGRIP sur la population virale et l'évolution clinique de sujets souffrant de pharyngite chronique non bactérienne.

Study was conducted on 20 subjects with non bacterial chronic rhinopharyngitis. Forty-one virus strains, mainly adenoviruses, type 3 parainfluenza virus, influenza and type 1 herpes viruses, as well as enteroviruses were isolated from samples collected at regular intervals. After the NIVGRIP (R) vaccination, the number of subjects with viruses present in the pharynx decreased by 75% (from 20 to 5 positive patients) and that of isolates from 41 to 6 (80% positive samples before vaccination against 10% after immunization).

Effect of the oral administration of NIVGRIP inactivated influenza vaccine on virus carriage in the nasopharynx of children aged 0-5 years.

The NIVGRIP inactivated influenza vaccine was administered by oral route to 11 children of a semi-closed pre-school community, found to carry viruses in their nasopharynx. Periodic virological investigations demonstrated that after vaccination virus carriage disappeared completely in 7 children; in the remaining 4 cases there was a considerable decrease (from 14 to 6) in the number of virus strains isolated. The virus isolates obtained after vaccination were represented exclusively by enteroviruses (Coxsackie B1 and B3, poliovirus type 1), in contrast with the wider range of viral agents (adeno-, parainfluenza, herpes, Coxsackie, and polioviruses) detected prior to vaccine administration.

Dynamics of neutralizing secretory influenza antibodies after nasal administration of the NIVGRIP trivalent influenza vaccine.

The trivalent inactivated NIVGRIP influenza vaccine was administered by nasal route to 26 young adults; a matched control group was represented by 19 subjects who received a placebo. Vaccination was followed by a higher proportion of rises in the level of neutralizing secretory antibodies (70-77%) than in that of serum HAI antibodies (19-42%) to the three influenza strains contained in the vaccine. Simultaneous rises in secretory antibody titers to three and two influenza antigens occurred in 50% and 23% of the vaccinees, respectively. The vaccine conferred a protection rate of 75%, as estimated by a 7-month clinical follow-up.

Viral and rickettsial etiology of an outbreak of acute respiratory tract infection.

An outbreak of 111 cases of acute respiratory tract infection was recorded in a community of the town "T" in April-May 1984. The clinical picture was severer than usual; 28% of the cases had to be hospitalized, average absenteeism being as high as 26 days per case. Serological investigations demonstrated the previous circulation of influenza virus B/Singapore/222/79 and the simultaneous circulation during the outbreak of influenza virus A/England/333/80 (H1N1) and of Rickettsia burneti (as also ascertained by isolation in the chick embryo of the former and by visualization by immunofluorescence in exfoliated cells of the latter pathogen). The association of the two etiological agents appears to account for the severe and protracted course of the disease.

Persistence of viruses in the nasopharynx of apparently healthy children aged 0-5 years. Results of investigations performed in 1982-83.

Virus isolation attempts were made with nasopharyngeal secretions from 400 apparently healthy 0-5-year-old children of a semi-closed community. Repeated virus isolations were achieved in 25 cases subjected to periodic investigations in 1982-83. One-two reisolation(s), 1-5 months apart, of the initially detected viral agents could be obtained in 15 out of the 25 children. The following viruses were reisolated (in decreasing order of frequency): adenoviruses, respiratory syncytial virus, Coxsackie B1 virus, herpes virus type 1, parainfluenza virus type 3. The problems raised by the carriage of respiratory viruses in the nasopharynx of apparently healthy children are discussed.

Efficiency of the NIVGRIP inactivated influenza vaccine applied by oral route: evaluation in a community of pre-school children in the period 1981-84.

The NIVGRIP inactivated influenza vaccine prepared in the "Stefan S. Nicolau" Institute of Virology was administered by oral route to 3-6-year-old children of a preschool community in October 1981, 1982 and 1983. There were no cases of acute respiratory tract infection (ARTI) in the community in the winter seasons of 1981-82, 1982-83 and 1983-84, with the exception of 3 ARTI cases recorded in February 1982 in unvaccinated children. In a similar community where no vaccination had been performed morbidity by ARTI was of 20% in 1982-83 and 12.6% in 1983-84. The efficiency and the advantages of oral influenza vaccination are outlined.

The effect of influenza vaccination on the circulation of viruses in the respiratory tract of apparently healthy schoolchildren aged 7 to 14 years.

Virus isolation attempts were made with 500 nasopharyngeal secretion samples from apparently healthy schoolchildren aged 7-14 years, vaccinated by nasal route with an inactivated influenza vaccine and with 500 samples from unvaccinated schoolchildren of the same age. The number of virus isolates obtained was much smaller in the former than in the latter group (13 as against 50 virus isolates). The role of influenza vaccination in the protection of schoolchildren against respiratory tract infections is discussed.

Comparative results of respiratory virus isolation attempts in chorioallantoic membrane fragments and embryonated chicken eggs.

Attempts performed with 39 samples of nasopharyngeal secretion resulted in the isolation of 21 (54%) hemagglutinating viral agents in chorioallantoic membrane (CAM) fragments, while a single strain (2.5%) was isolated in the embryonated egg. The time interval required for virus isolation in CAM fragments ranged from 4 to 20-24 days and was longer then 10 days in the embryonated egg. Maintenance of CAM fragments in roller tubes leads to a more abundant virus multiplication and, implicitly, to a reduction of the time interval necessary for virus isolation.

Investigations on the circulation of viruses occurring in the respiratory tract of apparently healthy schoolchildren aged 7 to 14 years.

A number of 92 viral agents could be isolated from 1,000 nasopharyngeal secretion samples collected from apparently healthy schoolchildren aged 7-14 years. The following viruses were identified, in decreasing order of frequency: 42 influenza A/USSR 90/77 (H1N1), 20 adeno-, 20 parainfluenza, 8 Coxsackie B1 and 2 herpes virus strains.

Isolation of parainfluenza virus type 3 from an infant with meningoencephalitis.

A parainfluenza virus type 3 strain was isolated from the cerebrospinal fluid of an infant with a clinical diagnosis of meningoencephalitis. Specific HAI antibodies to parainfluenza virus type 3, ranging in titer from 1/80 to 1/160 could be detected in the infant's serum. A 2-fold rise in the level of complement fixing serum antibodies-from 1/16 at the first to 1/32 at the second collection-was recorded.

Extrarespiratory localizations of parainfluenza viruses.

Two hemagglutinating and hemadsorbant agents-serologically identified as parainfluenza viruses type 3-were isolated from the cerebrospinal fluid of a 10-month-old infant with meningoencephalitis and from the urethral secretion of a male patient with nonbacterial urethritis. Parainfluenza virus antigens types 1, 2, and 3 were detected by indirect immunofluorescence reactions in cells exfoliated in the vagina of women with genital neoplasia or common gynecopathies.

Research on respiratory virus diseases performed in the "Stefan S. Nicolau" Institute of Virology.

The studies on respiratory virus diseases performed for 30 years in the "Stefan S. Nicolau" Institute of Virology approached various aspects of the ecology of inlfuenza and parainfluenza viruses, adenoviruses and other respiratory viruses, including numerous experimental, fundamental, clinical and morphopathological investigations. Special attention was paid to specific influenza prophylaxis and to the preparation of an inactivated influenza vaccine applicable by nasal and oral route.

Neutralizing influenza antibodies, IgA and total protein in the nasopharyngeal secretions of subjects vaccinated by nasal route with the inactivated influenza vaccine prepared in the "Stefan S. Nicolau" Institute of Virology.

Intranasal administration of two doses of the inactivated influenza vaccine prepared in the "Stefan S. Nicolau" Institute of Virology was followed by rises in the level of neutralizing secretory influenza antibodies in 82% of the cases. The concomitant study of secretory antibody, IgA and total protein levels, as well as of the serum HAI influenza antibodies demonstrated that their evolution was parallel only in 23% of the vaccinees. The percentage of secretory antibody conversion was similar to the rate of protection conferred by the vaccine.

The experience of the "Stefan S. Nicolau" Institue of Virology in the preparation of administration of inactivated influenza vaccines applicable by nasal or oral route.

Studies conducted for over 20 years allowed the authors to accumulate a rich experience in the production and administration of an inactivated influenza vaccine applicable by nasal and oral route, prepared in the "Stefan S. Nicolau" Institute of Virology. Applied as a monovalent in a single dose of 1000 IU, the vaccine induced seroconversion of 70% of the vaccines and ensured a 2--4-fold decrease in influenza morbidity. Owing to these qualities the vaccine represents one of the most advantageous preparations used in influenza prophylaxis.