Pentti Ukkonen

Cytomegalovirus Is Frequently Isolated in Bronchoalveolar Lavage Fluid of Bone Marrow Transplant Recipients without Pneumonia

We prospectively studied the presence of cytomegalovirus in bronchoalveolar lavage through virus isolation in 21 bone marrow transplant recipients before and after transplantation. All 14 lavage specimens collected before bone marrow transplantation were negative for cytomegalovirus. During the period from 20 to 90 days after transplantation, 12 of 24 lavage specimens (50%) from 14 patients without lung problems were positive for cytomegalovirus. Cytomegalovirus was isolated from 4 of 10 lavage specimens (40%) in 7 patients with pneumonia during the same interval. We conclude that culture for cytomegalovirus in bronchoalveolar lavage fluid is not a reliable method for establishing the viruss causative role in pneumonia soon after bone marrow transplantation. Among 14 patients in whom bronchoalveolar lavage was done at an asymptomatic stage, 6 of 9 patients with cytomegalovirus and none of 5 without cytomegalovirus in the lavage fluid later developed pneumonia. All of the patients subsequently developing pneumonia also had acute graft-versus-host disease, suggesting an immunopathologic mechanism, possibly triggered by cytomegalovirus, for cytomegalovirus-associated pneumonia.

Rubella immunity and morbidity: impact of different vaccination programs in Finland 1979-1992.

As the previous vaccination of 11- to 13-year-old girls proved ineffective, nationwide vaccination of preschool children with 2 doses of combined vaccine against measles, mumps, and rubella was started in Finland in 1982. To study the impact of vaccination, age-stratified rubella immunity and the occurrence of serologically verified rubella cases were determined using the computerized data of our diagnostic virus laboratory. The analysis covered the period 1979-1992, included all ages, and was based on the test results from 94,000 sera. By 1992, the seropositivity rate was 92-100% in 2- to 15-year-old children, remained high in females of all ages, but showed a gap in 16- to 19-year-old males. The number of verified rubella cases decreased to about 1/100, but outbreaks still occurred until 1991, when most cases were among adolescent males. The better protection of women was due to the vaccination of prepubertal girls since 1975. No congenital rubella infections were diagnosed after 1986. The 2-dose immunization of preschool children, complemented by selective vaccination of certain other groups, has resulted in excellent immunity in children and young adults, and practically eliminated rubella from the country.

Virus Inactivation during Intravenous Immunoglobulin Production

Effects of time, temperature, pH and stabilizers (i.e. medium) on inactivation of lipid‐enveloped model viruses, Semliki Forest and vesicular stomatitis viruses in the production process of intravenous immunoglobulin were investigated on a laboratory scale. The lowering of pH, the raising of temperature and the increasing of incubation time improved the inactivation effect. However, small changes in pH and stabilizer concentrations did not influence the results. Inactivation was not linear and a clear tailing off could be seen. Therefore, for complete virus inactivation incubation times longer than 20 h are necessary. Inactivation took place much more rapidly in intravenous immunoglobulin solution than in intramuscular immunoglobulin solution. Processing steps such as freeze‐dying in the presence of ethanol or storage of intramuscular immunoglobulin in the liquid state at pH 7 only partially inactivated these viruses.

Rubella Immunity and Morbidity: Effects of Vaccination in Finland

The occurrence of rubella antibodies and frequency of virologically proven rubella infections in different age groups were analyzed in a large serum material (about 60,000 sera) collected both before and after the start of nationwide vaccination of children against measles, mumps and rubella in Finland in 1982. The combined live vaccine significantly raised the rubella immunity of children and shifted rubella infections to older ages. In 1986, 91-98% immunity was found in the 2-10-year-old children so far covered by the vaccination programme; no rubella cases were diagnosed in this age group. We also demonstrated that another rubella vaccine given to about 60% of 13-year-old girls since 1975 both raised the immunity and reduced the occurrence of rubella in the vaccinated population. It is concluded that the rubella vaccinations, especially the combined vaccine given to small children, are effective, although the total number of reported rubella cases in the whole population did not decrease significantly during the study period.

Multiple structurally related defective-interfering RNAs formed during undiluted passages of Semliki forest virus.

Abstract Semliki Forest virus (SFV) was transferred serially with undiluted inoculum for 24 successive passages in BHK cells and the virus-specific RNAs were isolated from infected cells and from virus particles released into the medium. The infectivity and hemagglutination titers started to drop after 4 serial passages. A new virus-specific intracellular RNA species sedimenting at 18 S appeared in the 4th passage and remained the dominant DI-RNA up to the 17th passage. The 18 S RNA population was incorporated very inefficiently into viral particles, but was replicated more rapidly than other viral RNAs. Another DI-RNA, sedimenting at 24 S, was found in virus particles starting at the 4th passage. The 24 S RNA replicated less efficiently but was incorporated in virus particles 3 to 5 times more efficiently than the 18 S RNAs. After 18 undiluted passages a switch in the distribution of intracellular RNAs resulted in the predominance of a 24 S RNA species. The early and late 24 S RNA species were found to be related but not identical when compared by ribonuclease Tl oligonucleotide mapping. The results indicated that the early 24 S RNA fingerprint closely resembled that of the 18 S DI-RNA population, whereas the late 24 S RNA had a lower complexity. A 33 S DI-RNA species isolated from virus particles at the 21st passage also had a simple T1 oligonucleotide pattern indistinguishable from that of the late 24 S RNA. These results suggest that the late 24 S DI-RNA molecules contain multiple repeated sequences similar to those recently found in the 18 S DI-RNA.

The seroepidemiology of Chlamydiae in Finland over the period 1971 to 1987.

The seroepidemiology of chlamydial infections in the Finnish population was studied by analysing the prevalence of chlamydial complement fixing (CF) antibodies in patients sera sent for virus serological screening tests over 17 years from 1971 to 1987. The total number of sera studied was over 160,000. In the early 1970s, the prevalence of chlamydial CF antibodies (CF titres greater than or equal to 8) was low (less than 2%), but later the proportion of seropositive cases rose, and in 1976, 18% of sera contained antibodies. In 1984, the seropositivity rate was over 31%. The prevalence of high chlamydial CF titres (titres greater than or equal to 64) also showed annual variation. In general, under 1% of sera contained chlamydial CF antibodies in high titre, but in 1979 and 1984, distinct peaks occurred when 1.3% and 1.4% of sera, respectively, had titres greater than or equal to 64. The age-related antibody positivity rate showed a decline during early infancy, an increase in childhood and adolescence, and a stable level in adulthood when approximately 20% of the sera contained antibodies. The chlamydial antigen used in this survey was genus-specific, i.e. it detects antibodies against all chlamydial species. Epidemiological data support the hypothesis that infections due to a novel chlamydial species, TWAR chlamydia, are the most likely explanation for the relatively frequent occurrence of chlamydial CF antibodies and for the variation in CF antibody prevalence.

Solid-phase enzyme immunoassay for rotavirus antigen: Faecal protease activity as a reason for false-negative results

Rabbits and guinea pigs were immunized with purified bovine rotavirus. Immunoglobulin G fractions of the resulting antisera were used in a standard four-layer solid-phase enzyme immunoassay (EIA) for rotavirus antigen in human faecal specimens. Samples negative for rotavirus in electron microscopy, when diluted in standard EIA buffers, regularly gave absorbance values lower than those obtained with buffer blank only. By further diluting of the samples the resulting absorbance values were found to increase to the blank levels. When all dilution buffers were supplemented with 1-5% of bovine serum, negative samples at any dilution gave absorbance values close to those of the buffer blanks. Similar results were obtained if the serum was replaced by 1-5 mM of phenylmethylsulphonyl fluoride, a synthetic broad spectrum serine-type protease inhibitor. Aprotinin, another protease inhibitor, was without effect. A similar inhibition pattern was obtained when faecal specimens were tested in a caseinolytic quantitative protease assay in the presence of the above inhibitors. These observations suggest that protease activity present in human faecal samples may cause false-negative results in solid-phase immunoassay for viral antigens, unless appropriate means are used to avoid this interference.

Proportions of immunoglobulin isotypes in paralytic poliomyelitis and after vaccination

Immunoglobulin isotype composition of poliovirus antibodies was studied by isotype-specific solid-phase radio-immunoassay (RIA) in four patients with paralytic poliomyelitis, five adults receiving live poliovirus vaccine as a booster immunization, and seven children receiving first doses of inactivated poliovirus vaccine. In paralytic poliomyelitis serum and cerebrospinal flind (CSF) poliovirus antibodies were mainly of IgG1, IgG3, and IgA isotypes. IgM antibodies were found in sera but not in CSF. Either IgG2 and IgG4 antibodies were undetectable or the titers were low. In adults who had received live trivalent poliovirus vaccine, antibodies against poliovirus type 3 were detected in IgG1 (53% of total antibodies), IgG3 (25%), IgM (9%), IgA (8%), IgG2 (3%), and IgG4 (2%) isotypes. In prevaccination and late postvaccination sera the share of IgG3 antibodies was exceptionally high (35%). In children who received inactivated poliovirus vaccine, antibodies developed in IgG1 (53–61% of total antibodies for poliovirus types 1, 2, and 3), IgG3 (12–21%), and IgM (23–33%) isotypes. Antibody levels in IgG2, IgG4, and IgA isotypes were low and observed only in a few cases. Like other viral antibodies IgG1 and IgG3 isotypes were the major IgG subclasses in poliovirus antibodies.

Preparation of nasopharyngeal secretions for immunofluorescence by one-step centrifugation through Percoll

A simple method was developed for separation of cells from nasopharyngeal secretion for the diagnosis of respiratory virus infections by immunofluorescence microscopy. The diluted specimen, containing dithiothreitol to break up the mucus, was centrifuged once through a cushion of 20% Percoll (colloidal silica, a density gradient medium), which permitted sedimentation of cells through the cushion, but retained mucus on top of it. The pelleted cells were resuspended, and microscope slides were then prepared by standard techniques. The Percoll centrifugation method was also applicable for sputum and bronchoaveolar lavation specimens. Immunofluorescent antibody staining of nasopharyngeal secretions prepared by the described method was more sensitive than enzyme immunoassay for the detection of respiratory syncytial virus.

RSV infection complicating the therapy of pediatric malignancies: Report of six cases

We describe a series of six patients with symptomatic respiratory syncytial virus (RSV) infections while receiving anticancer chemotherapy. Particularly during epidemics in the general population, RSV remains a potential cause for morbidity and even mortality among children immunocompromised through the administration of anticancer chemotherapy and especially those being transplanted. We emphasize the importance of rapid diagnostics as well as prevention of the spread of the virus in a pediatric hematology/oncology unit.