Diagnosis of Mycoplasma pneumoniae pneumonia: sensitivities and specificities of serology with lipid antigen and isolation of the organism on soy peptone medium for identification of infections.

The sensitivities and specificities of isolation and serology for detection of Mycoplasma pneumoniae infections were determined for 3,546 pneumonia patients for whom both isolation and serological data were available. Soy peptone, fresh yeast extract, horse serum-supplemented agar, and diphasic medium were employed for isolation, and the lipid antigen of M. pneumoniae was used for serodiagnosis by complement fixation. The number of M. pneumoniae colonies most frequently detected was 200 to 600 per throat swab, with a range of less than or equal to 60 to greater than or equal to 2,000. The use of diphasic medium increased the number of isolates by 26% compared with direct isolation on agar plates. The organism was isolated from 360 of 525 patients who showed fourfold or greater antibody increases in their paired sera, resulting in a sensitivity of culture of 68%. When persons with titers of greater than or equal to 32 but no fourfold increase were used as the reference, the sensitivity of culture was 58%. The combined sensitivity of the culture method for persons with serological evidence of infection (fourfold increase and titer of greater than or equal to 32) was 64%. The specificity of the culture method was 97% for the 2,527 serologically negative persons. Fourfold antibody increases were found in 360 of 674 persons with isolates of the organism, resulting in a sensitivity of 53%. An additional 247 persons showed titers of greater than or equal to 32 (without a fourfold increase), resulting in a combined sensitivity of 90% for serology with the lipid antigen for the detection of antibodies in culture-positive persons. Fourfold antibody increases were found in 6% of culture-negative persons, resulting in a specificity of 94%. The quantitative culture results provide important base-line data for the development of rapid diagnostic tests for M. pneumoniae infection.

Animal exposures and antibodies to Toxoplasma gondii in a university population.

To determine the risk of toxoplasma infection to individuals exposed to cats in a research institution, we compared the prevalence of toxoplasma antibodies with exposure to cats in university employees. Of 116 employees tested, 42 (36 percent) had toxoplasma antibodies as determined by the indirect fluorescent antibody test. Women and individuals aged 35 years or more had a greater prevalence of antibodies. The antibody prevalence by occupation was 72.1 percent for physicians and those with doctorates, 45.3 percent for animal and veterinary technicians, 33.3 percent for research technicians, 28.2 percent for administrative staff, 25.0 percent for graduate students and fellows, and 13.4 percent for veterinarians. There was no significant positive association between exposure to cats and the prevalence of toxoplasma antibodies. A follow-up of seronegative employees, 6 and 18 months later, revealed no seroconversions indicative of acute toxoplasma infection. We concluded that there was no significant risk of toxoplasma infection in university employees exposed to cats.

The effect of respiratory viral infections on patients with cystic fibrosis.

We examined, in a 2-year prospective study, the frequency of respiratory viral infections in 19 school-age patients with cystic fibrosis and their unaffected siblings. At 2-month intervals throughout the study period, pulmonary function tests, oropharyngeal cultures, and serologic tests for respiratory viruses were performed in all subjects. Quantitative sputum cultures for bacteria were performed in subjects with cystic fibrosis. The same laboratory specimens were also collected at the time of all acute respiratory illnesses. Over the 2-year period, 398 viral cultures and serum samples were collected, 210 from patients with cystic fibrosis and 188 from their siblings. The frequency of culture-documented and seropositive viral infections was not significantly different between patients with cystic fibrosis and their siblings. The patients with the highest frequency of viral infection were younger and had the lowest rate of decline in lung function and severity score. We conclude that school-age patients with cystic fibrosis are no more susceptible to viral infections than their unaffected siblings. We were unable to demonstrate any significant adverse effect of respiratory viral infections on pulmonary function in 19 patients with cystic fibrosis aged 5 to 21 years.

Persistent enteral infections with adenovirus types 1 and 2 in infants: no evidence of reinfection.

Isolates of adenovirus types 1 and 2, obtained from 11 infants with prolonged faecal excretion (up to 515 days), were compared by DNA restriction analysis with seven standard endonucleases which recognize hexanucleotides and two additional endonucleases which recognize tetranucleotides. In all instances identical genome types were identified in isolates obtained early and late after infection. Our interpretation of these data is that a chronic persistent infection occurred in these children, and not a reinfection with the same serotype.

Human rhinovirus 3C protease: cloning and expression of an active form in Escherichia coli.

A cDNA encoding the viral protease from the 3C region of human rhinovirus type 14 was expressed in Escherichia coli through the use of a periplasmic secretion vector. The recombinant protease contained an eight amino acid N-terminal extension that enabled its detection by a specific antibody. It was expressed at a level of approximately 1 mg/L of E. coli culture. Biological activity of the protease was assessed in vitro by using a chemically synthesized peptide consisting of a consensus picornavirus protease cleavage site, Arg-Ala-Glu-Leu-Gln-Gly-Pro-Tyr-Asp-Glu. The peptide was cleaved by the recombinant protease at the Gln-Gly bond, generating the product peptides Arg-Ala-Glu-Leu-Gln and Gly-Pro-Tyr-Asp-Glu, which could be separated from the substrate peptide by reversed-phase HPLC. An in vitro assay for the rhinovirus 3C protease was developed by observing the rate of disappearance of the substrate peak from chromatograms of the supernatants of digestion mixtures.

Influenza B in households: virus shedding without symptoms or antibody response.

In the fall of 1983, 53 households were enrolled in a double-blind trial of alpha 2-interferon as an intranasal spray to prevent common colds. During the winter/spring of 1984, 26 households were infected with influenza type B, as shown by isolation of the virus (19 households) and/or significant antibody titer rises (seven households). Interferon did not prevent influenza B infection or modify resulting illness. Of 37 persons shedding virus, 12 were asymptomatic. All were older than age 12 years, and 10 did not respond with antibody by any of the five test methods employed (complement fixation, hemagglutination inhibition, enzyme-linked immunosorbent assay (ELISA), neutralization, and Western blot). In contrast, of 13 symptomatic persons shedding virus from whom sera were available, 11 had significant antibody titer rises. Infection rates were highest among teenagers, but also surprisingly high among the 11 persons observed who were aged 50 years or older, four of whom were infected. The case-to-case interval in household transmission varied between one and nine days. Longer intervals of one, two, and four months between infections among family members were also observed, suggesting repeated introductions. Neither virus isolation alone nor serologic tests was sufficient to estimate infection rates.

A collaborative report: rhinoviruses--extension of the numbering system from 89 to 100.

To define the number of rhinovirus serotypes, cross neutralization tests and characterization studies were completed on 25 candidate prototype rhinoviruses submitted to a third phase of a collaborative program. Based on the results, 11 distinct prototype strains were designated and the numbering system was extended to include 100 rhinoviruses. In addition, recent evidence indicates that over 90% of rhinoviruses isolated in three areas of the country could be typed with antisera for rhinovirus types 1-89.

Use of bacterial antigen detection in the diagnosis of pediatric lower respiratory tract infections.

Two immunochemical methods were used to identify Haemophilus influenzae and Streptococcus pneumoniae capsular antigens in the urine and serum of 162 children with acute lower respiratory tract infection. These methods were compared with standard bacterial blood culture. Viral and mycoplasma cultures of respiratory secretions were obtained simultaneously to determine the frequency of antigenuria at the time of nonbacterial acute lower respiratory tract infection. Urine from groups of well children and children with acute otitis media was tested for capsular antigens to determine the incidence of antigenuria. Antigenuria was found in 24% of children 2 months to 18 years of age with acute lower respiratory tract infection compared with a 2% incidence of bacteremia. Antigenuria was found in 4% of asymptomatic children and 16% of children with acute otitis media. One third of children with symptoms of acute lower respiratory tract infection and viral isolates from the oropharynx had bacterial antigenuria. The sixfold increase in frequency of bacterial antigenuria in children at the time of lower respiratory symptoms suggests that bacterial acute lower respiratory tract infection may be more common than identified by traditional culture techniques. Because bacterial antigen may come from other sites such as the middle ear, further studies are needed to determine the role of antigen detection in the diagnosis of pediatric acute lower respiratory tract infection.

Rises in titers of antibody to human coronaviruses OC43 and 229E in Seattle families during 1975-1979.

Sequential serum specimens were obtained every four months during 1975-1979 from 44 children and adults of 10 Seattle families. The 419 specimens were tested for antibody to human coronaviruses OC43 and 229E by enzyme-linked immunosorbent assay (ELISA). Antibody titers were found to increase with age, and titers as well as frequency of rises were greater for OC43 than for 229E virus in all age groups. Significant antibody rises were most frequent in specimens bracketing the winter interval, but some also occurred in the spring-summer and summer-fall intervals. Concurrent significant antibody rises to OC43 virus in different members of the same family were observed in 15 instances, to 229E virus in seven instances, and to OC43 virus in some members and 229E virus in others in eight instances. Significant antibody rises to OC43 or 229E virus indicating reinfections were frequently observed throughout the three-year period but were always separated by at least two four-month intervals. Concurrent significant antibody rises to both 229E and OC43 viruses were seen only in three persons. Finally, the frequency of significant antibody rises in children, about one per person-year, was almost three times higher than in adults.

Rhinoviruses in Seattle families, 1975-1979.

Rhinovirus infections in Seattle families with schoolchildren (1975-1979) and in selected outpatients were revealed by virus shedding or antibody rise. These observations extend those in the Seattle Virus Watch (1965-1969). Analysis of rhinovirus serotype prevalence again revealed certain "common" persisting serotypes but provided no further evidence that new serotypes are continuing to emerge. Two seasonal peaks, spring higher than fall, were again evident. Infection rates, again inversely related to age, were lower overall than in the Virus Watch (0.42 vs. 0.64 per person-year), probably because there were fewer young children. Frequencies of antibody response by virus shedders again varied widely by serotype but differed greatly from those in the Virus Watch in rank order of response rate, suggesting that immunogenicity is not a stable serotype characteristic. The frequency and magnitude of antibody response of virus shedders increased with age. Antibody-related protection against infection was evident only in persons age greater than or equal to 10 years. Observations in 7 families during successive homotypic infection episodes indicate that postinfection immunity to natural challenge requires persistence of antibody. Of all reported respiratory illness, 11.9% (0.31 per person-year) were due to rhinoviruses and 6.9% to influenza viruses. Of viruses recovered from family members, rhinoviruses, herpes simplex, and influenza comprised 56%, 12.6%, and 12.4%, respectively. Although households often experienced greater than or equal to 2 concurrent or closely consecutive episodes of infection with different viruses, only 29 individuals were shown to shed 2 viruses at the same time. Most of the second viruses, include 3 rhinoviruses and 18 other nonhemadsorbing viruses, appeared when 582 rhinovirus-positive specimens were retested after treatment with homotypic antibody. These results suggest that rhinoviruses interfere with nonhemadsorbing viruses in cell culture but mostly with other rhinoviruses in humans.

Molecular epidemiology of adenoviruses: global distribution of adenovirus 7 genome types.

Adenovirus 7 (Ad7) is the adenovirus species that most frequently has been associated with severe illness. Seven distinct genome types of adenovirus 7, Ad7p, Ad7a, Ad7b, Ad7c, Ad7d, Ad7e, and Ad7f, can be identified by using restriction endonucleases BamHI and SmaI. We analyzed the distribution of the different Ad7 genome types among 314 isolates from patients and healthy shedders. The Ad7b and Ad7c genome types accounted for 90% of the isolates from patients and appeared to be mutually exclusive. A shift from Ad7c to Ad7b genome types occurred in 1969 in Europe and in 1975 in Australia. During the last decade, Ad7b genome types predominated in Australia, Europe, and North America. Ad7c was detected in South Africa, Ad7d was detected in China, Ad7e was detected in Brazil, and Ad7f was detected in Australia. The Ad7p and Ad7a genome types dominated among isolates obtained from healthy shedders and appeared scattered through the years and the geographical areas. The prevalence of Ad7 infections is high in Japan as judged by the herd immunity. However, the low percentage (2%) of Ad7 isolates among all adenovirus isolates chiefly from patients, coupled with 30 to 50% antibody prevalence, argues for a high proportion of inapparent infections and, hence, Ad7 strain(s) of low pathogenicity.

Microbial causes of neonatal conjunctivitis.

We assessed the microbial causes of neonatal conjunctivitis by comparing 55 infants with purulent conjunctivitis and 60 healthy control infants. A mean of greater than 5 leukocytes per 1000X microscopic field was seen in Gram-stained smears obtained from the more inflamed eye in 77% of 30 untreated patients but none of 57 controls. Pathogens isolated more often from untreated patients than from controls included Haemophilus spp. (17% vs 2%, P = 0.01), Staphylococcus aureus (17% vs 2%, P = 0.01), Chlamydia trachomatis (14% vs 0%, P = 0.01), enterococci (8% vs 0%, P = 0.05), and Streptococcus pneumoniae (11% vs 2%, P = 0.06). One or more of these pathogens were isolated from the conjunctivae in 58% of patients and 5% of controls (P less than 0.001). Bacterial morphology seen on smear correlated with the pathogens cultured. Isolation of Haemophilus spp. or S. pneumoniae was associated with dacryostenosis. We conclude that several microbial pathogens are implicated in neonatal conjunctivitis. These organisms have differing susceptibilities to antimicrobial agents, so culture and sensitivity testing are required as a guide to therapy.

Influenza surveillance in the Pacific Northwest 1976-1980.

Between June 1976 and June 1980 active year-round surveillance for influenza was carried out in Seattle in order to establish an early warning system. This report compares yield by different community groups and age. Waves of influenza virus infection appearing in three successive springs were followed in each instance by epidemics with the same subtype virus(es) in the following winter. These included two co-circulating A/H3N2 variants (A/Victoria/75 and A/Texas/77) in spring 1977 and winter 1977-1978, A/H1N1 (A/USSR) in spring 1978 and H1N1 (A/Brazil) winter 1978-1979, and type B influenza in spring 1979 and winter 1979-1980. Despite intensive surveillance through the summer and fall, the first isolate was not obtained until early December each year. Young adults (18-30) were as good sources for influenza viruses as children (less than 18).

Naturally acquired immunity to pneumonia due to Mycoplasma pneumoniae.

The immunity to Mycoplasma pneumoniae was studied by periodic collection of sera up to 15 years after infection. Sera were tested for antilipid antibodies by complement fixation. Antibody levels remained elevated for two to nine years after pneumonia but usually fell sharply after the second year in persons with milder symptoms. Infection rates were at least six times higher in comparison groups than in previous pneumonia patients. Schoolchildren with serologic evidence of infection, but without pneumonia, during the 1966-1967 epidemic were not protected during the next epidemic (1974). Children with evidence of infection during the first two years of life were at no higher risk of clinical pneumonia (immunopathological response) at school age than those without previous known exposure. The current study suggests that naturally acquired infection induces partial immunity which lasts longer after pneumonia than after mild infections.

Isolation of rhinovirus intertypes related to either rhinoviruses 12 and 78 or 36 and 58.

Many antigenic relationships have been demonstrated among the 90 rhinovirus serotypes. Among these are reciprocal cross-reactions between serotypes 12 and 78 and between serotypes 36 and 58. Neutralizing-antibody titers to homologous virus of the related pairs are generally 16- to 64-fold higher than to the heterologous member, and neutralization by heterologous antiserum in the pools is not seen with prototype viruses. However, a number of isolates were encountered which gave anomolous results when tested with the antiserum pools in fetal tonsil cells. When these strains were tested in fetal tonsil cells against the monospecific antisera composing the pools, it was shown that several isolates were apparently intertypes, neutralized equally by antisera to related types 12 and 78 or 36 and 58. Isolate 1104, an apparent intertype between serotypes 36 and 58, and isolate 9433, intermediate between serotypes 12 and 78, were selected to use as immunogens in rabbits. When tested in HeLa cells, antiserum prepared against isolate 1104 neutralized isolates 1104, 58, and 36 at titers of 1280, 640, and 40, respectively. The k values against isolates 1104, 58, and 36 were 356, 145, and 4, respectively, indicating a much closer relationship of isolate 1104 to type 58 than to type 36. Similar results were obtained with isolate 9433. The neutralizing-antibody titer of anti-9433 serum was 160 against both 9433 and type 78 and was 20 against type 12. The k values of anti-9433 serum against 9433, 78, and 12 were 161, 111, and 2, respectively, indicating that 9433 and 78 were nearly identical. However, the respective neutralizing-antibody titers of anti-78 serum to type 78 and isolate 9433 were 640 and 80, and the respective k values were 172 and 85, demonstrating some antigenic differences. The discovery of intertypes confirms the antigenic variation among rhinoviruses, and the intertypes may represent links in the evolution of types. These observations also demonstrate that isolates in first or second passage in diploid cells may display an antigenic profile different from that seen in HeLa cells at high HeLa cell passage level.

Isolation of respiratory syncytial and influenza viruses from the sputum of patients hospitalized with pneumonia.

Routinely collected sputum specimens from 100 adults hospitalized with pneumonia were frozen at -70 C until inoculation into Madin-Darby canine kidney, fetal tonsil, and esophageal epithelial cells. Six influenza A (H3N2) viruses, two respiratory syncytial viruses, three rhinoviruses, and nine herpes simplex viruses were recovered. Four patients with influenza virus and one with respiratory syncytial virus isolated had nosocomial pneumonia. Viral isolation from sputum specimens may aid the diagnosis of pneumonia of unclear etiology and merits further evaluation as a diagnostic tool and as an adjunct to influenza surveillance.

Influenzavirus infections in Seattle families, 1975-1979. I. Study design, methods and the occurrence of infections by time and age.

Intensive surveillance of Seattle, Washington, families with school-age children for influenzavirus infections during 1975-1979 encompassed 639 family- and 2732 person-seasons of observation, covering four influenzavirus epidemic seasons: type B (1975-1979), type A/H3N2 (1975-1976 and 1977-1978) and type A/H1N1 (1978-1979). Late spring "herald" waves of infection occurred in 1977 (A/H3N2), 1978 (A/H1N1) and 1979 (type B), the latter presaging an epidemic in 1979-1980. Out-of-season infections, recognized by serology only, included type B and A/H3N2 viruses in each summer and A/H1N1 virus in 1978. In epidemic seasons, infection rates were highest in children aged 5-9 years (A/H3N2) or in teenagers (A/H1N1 and type B). A/H1N1 virus caused the sharpest epidemic, with 31% of the population (but only 2% of adults) infected and 72% of households invaded in 1978-1979. These compare with infection rates of 17-24% overall and 6-13% of adults and the invasion of 38-53% of households observed in the type B and two A/H3N2 epidemics. Extended observation (largely serologic) of a cohort of 1965-1969 Virus Watch families for up to 14 years (including one three-year gap) indicated overall infection rates of 13.7 and 16.4 per 100 person-years with types B and A/H3N2 viruses, respectively, and rates of first and second reinfections of about 3 and 1 per 100 person-years, respectively, with each virus. Close surveillance in 1975-1979 revealed second family episodes of infection with each prevalent virus, 37 with A/H3N2, 15 with type B and 13 with A/H1N1 virus. Risk of infection in these episodes was related more to current hemagglutination-inhibiting titers than to experience (infected or not) in the initial episodes, with 67-100% reinfection when titers were low. Among younger (less than 20 years old) members, related illness was as frequent with reinfection as with initial infection.