Molecular epidemiology of Bordetella pertussis by pulsed-field gel electrophoresis profile: Cincinnati, 1989-1996.

Reported cases of pertussis have increased in the United States, with peaks occurring every few years. Bordetella pertussis isolates collected in Cincinnati from 1989 to 1996 were analyzed with pulsed-field gel electrophoresis (PFGE), to evaluate trends. Among 496 isolates, 30 PFGE profiles were identified; 32% were CYXXI-010, the profile that predominated each year. Eighteen profiles (198 strains) were identified in 1989-1992, 20 profiles (197 strains) were identified during the 1993 epidemic, and 11 profiles (101 strains) were identified in 1994-1996. From 1989 to 1996, among 42 patients, isolates from household members in 17 (89%) of 19 households had concordant PFGE profiles. There was no association between PFGE profile and seasonality, age, and hospitalization or pneumonia in infants <1 year old. The 1993 epidemic was associated primarily with an increased prevalence of PFGE profiles that circulated before and after 1993, which suggests that the epidemic was due to factors other than the emergence of a novel B. pertussis strain.

Molecular epidemiology of Bordetella pertussis by DNA fingerprinting with pulsed-filled gel electrophoresis, 1989-1996

Pertussis is an endemic disease in the United States of America, with epidemics occurring every three to four years. In Cincinnati, Bordetella pertussis isolates collected from 1989 to 1996 were analysed by genomic subtyping with pulsed-field gel electrophoresis (PFGE) to evaluate the B pertussis population before, during and after a large epidemic of epidemiologically relevant changes. Among the 496 B pertussis isolates, 31 PFGE profiles were identified; 32 percent of isolates were CYXXI-010 and this profile predominated in each year. Nineteen, 20 and 12 PFGE profiles were identified in the pre-epidemic period (n=198), during the epidemic (n = 197) and in the post-epidemic period (n = 101), resulting in genotypic diversities of 0.82, 0.83 and 0.76 respectively. From 1989 to 1996, among 19 households clusters of 42 patients, 17 (89 percent) households had concordant PFGE profiles among isolates from household members. There was no association between PFGE type and seasonality, age, hospitalisation or pneumonia in infants. The 1993 epidemic was primarily associated with increased prevalence of B pertussis PFGE profiles that circulated before and after the epidemic, suggesting increased susceptibility to pertussis rather than a novel strain as a cause of the outbreak.(Au)

Plant products as topical microbicide candidates: assessment of in vitro and in vivo activity against herpes simplex virus type 2.

There is considerable interest in developing topical microbicides; products to be used intravaginally by women for protection against sexually transmitted diseases. Many compounds derived from plants have been shown to have antimicrobial properties. We examined 19 such compounds in vitro by plaque reduction assay to determine their activity against a common sexually transmitted pathogen, herpes simplex virus type 2. Compounds with an ED50 < or = 7.0 mg/ml were tested for efficacy in vivo. Four compounds, carrageenan lambda type IV, cineole, curcumin, and eugenol, provided significant protection (P < 0.05) in a mouse model of intravaginal HSV-2 challenge. Eugenol, which provided the greatest protection in mice was also evaluated using the guinea pig model of genital HSV-2 infection where it also demonstrated significant protection. Based on these results, several plant-derived compounds appear to warrant further evaluation as potential microbicides.

Evaluation of the ImmunoCardSTAT! rotavirus assay for detection of group A rotavirus in fecal specimens.

Rapid detection of group A rotavirus was performed by using ImmunoCardStat! Rotavirus (ICS-RV) (which uses immunogold-based, horizontal-flow membrane technology), two commercial enzyme immunoassays (Premier Rotaclone and TestPack Rotavirus), and electron microscopy. A total of 249 stool specimens collected from children with gastroenteritis between February and April 1997 were tested. After resolution of 19 of the 22 discordant results by reverse transcription-PCR for group A rotavirus, ICS-RV detected 125 positives while Rotaclone and TestPack detected 127 and 129 positives, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value were 94.0, 100, 100, and 93.4% for ICS-RV; 95.5, 100, 100, and 95.0% for Rotaclone; and 97.0, 96.5, 97.0, and 96.5% for TestPack. ICS-RV was sensitive and specific and was relatively simple to perform and interpret.

Absence of rectal colonization with vancomycin-resistant enterococci among high-risk pediatric patients.

We prospectively surveyed for rectal colonization with vancomycin-resistant Enterococcus among 93 high-risk pediatric patients who were hospitalized at least 5 (median, 20) days. Fifty-two patients (56%) had enterococcal colonization; none had active infection with Enterococcus. All enterococci were vancomycin-susceptible (minimum inhibitory concentration < or =4 microg/mL). Associated exposures included recent antibiotics (50, 96%), surgical procedures (26, 58%), and immunosuppression (15, 29%).

Complicated parapneumonic effusions in children caused by penicillin-nonsusceptible Streptococcus pneumoniae.

OBJECTIVE: To describe the clinical characteristics of complicated parapneumonic effusions (CPE) in children caused by Streptococcus pneumoniae nonsusceptible to penicillin (PCN-N) and compare their clinical outcome with CPE caused by penicillin-susceptible (PCN-S) organisms. DESIGN: Children with parapneumonic effusions were identified retrospectively between July 1992 and June 1996. Charts of patients with CPE were reviewed for data obtained at the time of hospital admission. In addition, outpatient charts and/or the families of children with CPE caused by PCN-N S pneumoniae were reviewed to identify specific risk factors associated with PCN-N organisms. RESULTS: Sixty-four cases of CPE were identified during the 4-year period. In 26 cases a bacterial pathogen was recovered, with S pneumoniae accounting for 23 of these isolates (88%). Of the 23 S pneumoniae cases, 17 were PCN-S and 6 cases were nonsusceptible. Complicated parapneumonic effusions caused by PCN-N S pneumoniae occurred in significantly younger patients than CPE that were PCN-S (2.1 years vs 7.9 years). Nonsusceptible effusions also had a higher incidence of bacteremia than PCN-S effusions (100% vs 29%). There were no significant differences between the two groups for duration of chest tube drainage, febrile days, oxygen use, and hospital stay. CONCLUSION: CPE caused by PCN-N S pneumoniae is associated with a younger age and higher rate of bacteremia than CPE caused by PCN-S strains. However, there were no significant differences in outcome measures between patients infected with susceptible or nonsusceptible organisms.

Pneumococcal pleural empyemas in children.

Empyema rarely complicates pneumonia. In a 361-bed regional pediatric hospital, 50 pleural empyemas were identified from 1988 through 1994; 17 (34%) occurred in the last 12 months of this period, for which the incidence was 3.3 per 100,000 of the population aged < or = 18 years (P < .05, chi 2 test). A significant seasonal prevalence was observed: 50% of cases occurred in the winter (P < .001, chi 2 test). In contrast with the findings of previous studies, in which empyemas predominantly occurred in infants, the median age of our patients was 7 years; underlying illnesses were present in only 10%, and all had community-acquired disease. Eighty-two percent had chest tubes inserted, 56% required a thoracotomy with pleural decortication, and 2% had a lobectomy. There were no deaths. Streptococcus pneumoniae was isolated in 40% of the cases; specimens in 44% of the cases were sterile. None of the empyemas were associated with Staphylococcus aureus or Haemophilus influenzae type b, and only one was caused by group A streptococcus. Among 13 S. pneumoniae isolates, the rate of resistance to penicillin was 15%; to erythromycin, 15%; to chloramphenicol, 31%; and to cefotaxime, 23%. The penicillin-resistance rate among blood and cerebrospinal fluid pneumococcal isolates was 17% during 1993-1994. Drug-resistant S. pneumoniae is now a recognized cause of pleural empyemas in children.

Safety of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA to the lungs of nonhuman primates.

To define the toxicity of cystic fibrosis transmembrane conductance regulator gene (CFTR) gene therapy with a replication-deficient recombinant adenovirus (Av1Cf2) in a nonhuman primate model, 10(10) plaque forming units (pfu) were instilled directly through a bronchoscope into the right lung of 5 macaques, and a lower dose of 4 x 10(6) pfu was administered to the right lung of 1 macaque. One sham-treated control received phosphate-buffered saline (PBS). The macaques were evaluated sequentially by clinical examination, vital signs, weight, hematology, blood chemistry, chest radiography, pulse oximetry, and bronchoalveolar lavage (BAL) at baseline and 3-28 days post-treatment. After the period of observation, macaques were sacrificed for autopsy and histological examination. The macaques tolerated the experimental therapy clinically with no changes in body temperature, oxygen saturation, heart rate, body weight, or blood pressure. However, 1 macaque with visible evidence of aspiration at the time of initial bronchoscopy developed tachypnea with right lower lobe (RLL) pneumonia on chest radiograph and by histology. There were no changes in Hgb, Wbc, BUN, plasma electrolytes, bilirubin, or hepatic transaminases. In the macaques that received 10(10) pfu, there was a progressive increase in the number of CD8+ lymphocytes in BAL that was maximal at 28 days. Histological examination of the treated lungs of the high-dose macaques at 3 days showed marked peribronchial and perivascular cuffing by inflammatory cells and alveolar accumulation of neutrophils and macrophages. The alveolitis appeared to be resolving at 28 days, although the perivascular and peribronchial aggregates of mononuclear cells were still present. In the high-dose macaques, BAL interleukin-8 (IL-8) was increased at all time points (256-388 pg/ml versus 1-84 pg/ml at baseline and in control), whereas IL-1 beta was increased only at days 21 and 28 (341-852 pg/ml versus 30-92 pg/ml at baseline and in control). There were no increases in BAL cell counts, IL-1 beta or IL-8, and histological changes were mild in the macaque that received 4 x 10(6) pfu. Evaluation for Av1Cf2-derived human CFTR expression using RS-PCR demonstrated expression at 3, 10, and 21, but not 28 days in macaques treated with 10(10) pfu of Av1Cf2. In situ hybridization analysis demonstrated human CFTR mRNA in the alveolar regions of the lobes that received the vector at 10 and 21 days. There was no evidence of expression after treatment with 4 x 10(6) pfu. This study showed that high-dose adenoviral vector administration to the lung achieved CFTR gene transfer and expression but was associated with increased concentrations of cytokines in BAL and alveolar inflammation. A low dose, equivalent to the maximum clinical dose currently proposed for phase I trials in human subjects, was not associated with cellular or cytokine evidence of inflammation, and histological abnormalities were mild.

Containment of pertussis in the regional pediatric hospital during the Greater Cincinnati epidemic of 1993.

OBJECTIVE: To describe methods of preventing nosocomial pertussis in patients, employees, and visitors to a hospital during a communitywide epidemic in Greater Cincinnati. DESIGN: Six-month descriptive study of the methods, effectiveness, and cost of a program to prevent nosocomial pertussis. SETTING: Three hundred sixty-one bed, tertiary-care, university, pediatric hospital. RESULTS: We educated 3,764 hospital employees about pertussis. We evaluated 206 employees with respiratory illnesses, based on clinical presentation, pertussis exposure, and work setting. Eighty-seven had pertussis: 84 coughed for > or = 2 weeks (outbreak clinical case definition), 65 had paroxysms, 27 whooped, 22 had posttussive emesis, and 13 were positive by direct fluorescent antibody or culture for Bordetella pertussis. Seventy-nine employees were sent on 5-day furloughs. Six hundred twenty-two employees received 14 days of erythromycin (579) or trimethoprim-sulfamethoxazole (43). Symptomatic patients were identified at triage in the emergency department and placed in respiratory isolation. Suspect pertussis cases were admitted in respiratory isolation. Among 49 toddlers who were given erythromycin and managed in "coughing respiratory cohorts," eight had proven pertussis. Inpatients were restricted to assigned nursing units. Respiratory masks were required for those entering the test referral center, where more than 3,500 pertussis cultures were performed. Hospitalwide visitor restriction was enforced for those aged 14 years or younger and for those with respiratory symptoms. Only parents and guardians were permitted to visit the newborn intensive care unit. A child-care service managed 488 inpatient sibling visitors. Four symptomatic children in the employees' child-care center were excluded pending physician evaluation; one had pertussis. CONCLUSIONS: Control measures appeared effective. Pertussis occurred in 87 (2%) employees. Among 102 children hospitalized with pertussis, respiratory isolation was delayed in nine cases, and one case was nosocomial. Program expenses totalled $85,400. Adult booster immunization with acellular pertussis vaccine might represent the safest and least expensive strategy for preventing epidemic pertussis, and controlled trials of acellular pertussis vaccine in hospital employees are needed.

Development of a polymerase chain reaction assay to detect the presence of Streptococcus pneumoniae DNA.

In this study, we have developed a chemically sensitive and specific polymerase chain reaction (PCR) assay to detect the presence of Streptococcus pneumoniae genomic DNA. The target DNA sequence was a 322-base pair segment of the S. pneumoniae DNA polymerase I gene (pol I). PCR products of pure cultures of a set of pneumococcal serotypes commonly associated with human infection could be amplified in water and in blood cultures of clinical isolates containing S. pneumoniae. We were able to detect 2 fg of purified S. pneumoniae DNA. There were no false-positive reactions when the assay was performed on samples containing the following clinically encountered bacteria: Haemophilus influenzae type B, Neisseria meningitidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas spp. nontypeable H. influenzae, Staphylococcus aureus, coagulase-negative staphylococci, and Streptococcus pyogenes. The addition of EDTA and citrate-anticoagulated whole blood to the PCR reaction mixture inhibited the PCR assay, whereas the addition of lithium heparin, sodium heparin, and sodium polyanetholesulfonate-anticoagulated whole blood to PCR reaction mixture did not interfere with the ability to detect the presence of S. pneumoniae DNA.

The 1993 epidemic of pertussis in Cincinnati. Resurgence of disease in a highly immunized population of children.

BACKGROUND: In 1993 there was a resurgence of pertussis in the United States. Altogether, 6335 cases were reported, the most in 26 years. METHODS: Using active microbiologic surveillance, we investigated the epidemic of pertussis in Greater Cincinnati in 1993. The population of 1.7 million in this area is served by a single children's hospital and pertussis laboratory. We prospectively followed patients given a new diagnosis of pertussis in July through September 1993 to determine the characteristics of the epidemic. RESULTS: From 1979 to 1992, there was a cumulative total of 542 cases of pertussis. In 1993, 352 cases were diagnosed, an increase of 259 percent over the 1992 total. Sixty-three percent of the cases had positive cultures for Bordetella pertussis, 18 percent were positive on direct fluorescent-antibody testing only, and 19 percent were diagnosed clinically. The outbreak began in the suburbs during the summer and spread through Greater Cincinnati. Of 255 total cases diagnosed in July through September (195 excess cases over the maximal base-line level of 20 per month in the previous 14 years), 75 percent were in white patients and 67 percent of the patients had private insurance or paid for care out of pocket. In 1993, as compared with 1979 through 1992, there was a shift in incidence from younger infants to older children; the percentages of cases according to age group were as follows: 0 to 6 months, 53 percent from 1979 through 1992 and 35 percent in 1993 (P < 0.001); 7 months to 5 years, 33 percent and 43 percent (P < 0.002); 6 to 12 years, 5 percent and 11 percent (P < 0.001); and more than 12 years, 5 percent and 11 percent (P < 0.003). Immunization records revealed that 74 percent (75 of 101) of the children with pertussis who were 19 months to 12 years old had received four or five doses of the combined diphtheria-pertussis-tetanus (DPT) vaccine, and that 82 percent (103 of 126) of those 7 to 71 months old had received at least three doses of DPT vaccine. The whole-cell vaccines used came from both of the major manufacturers (Connaught Laboratories and Lederle Laboratories). Disease was not severe, but 80 of the 255 children (31 percent) given diagnoses during the three epidemic months were hospitalized. There were no deaths. CONCLUSIONS: Since the 1993 pertussis epidemic in Cincinnati occurred primarily among children who had been appropriately immunized, it is clear that the whole-cell pertussis vaccine failed to give full protection against the disease.

A comparison of diagnostic methods in adolescent girls with and without symptoms of Chlamydia urogenital infection.

OBJECTIVE: To evaluate the clinical utility of various diagnostic tests, two enzyme immunoassays and a chemiluminescent DNA probe were compared with cell culture (with monoclonal antibody confirmation) for the diagnosis of endocervical Chlamydia trachomatis infection. DESIGN: The clinical performance of four diagnostic methods for Chlamydia trachomatis urogenital infections were compared, using specimens generated from consecutive pelvic examinations. SETTING: Subjects were recruited from an urban adolescent clinic that provides primary and referral care. PARTICIPANTS: A total of 479 adolescent female subjects were enrolled. The order of sample collection was randomized. Subjects were stratified according to whether they were asymptomatic (n = 228) or symptomatic (n = 251). MEASUREMENTS AND RESULTS: Discrepant analysis was performed when culture was negative and nonculture technique was positive. The subject was considered to have chlamydia if culture was positive, or if one or more nonculture techniques, with that test's confirmatory assay, were positive (consensus-positive). Prevalence of chlamydia was 11.0% in the asymptomatic, and 20.7% in the symptomatic, group. Overall, 32.5% of the infected subjects were asymptomatic. Sensitivity of diagnostic methods varied from 52% to 80% in the asymptomatic subjects, compared with 65% to 81% in symptomatic subjects. Culture sensitivity was 75% to 80%. The specificities of all tests were 96% or greater. Accuracy of nonculture methods varied from 89.5% (DNA probe, symptomatic subjects) to 96.9% (enzyme immunoassay asymptomatic subjects). CONCLUSIONS: There are significant differences in symptomatic subjects when evaluating accuracy of test outcome, using a consensus-positive criterion. Asymptomatic infections account for nearly one third of adolescent females infected with chlamydia. The prevalence of chlamydia urogenital infections are underestimated by any single diagnostic test, particularly in the asymptomatic patient.

Tracheal granulation tissue. A study of bacteriology.

We prospectively examined 19 patients (21 laryngotracheal reconstructions) over a 6-month period to evaluate the bacteriology of granulation tissue present at the time of Teflon stent removal and at the first laryngoscopy several weeks later. The most frequently recovered isolates were viridans streptococci, Pseudomonas aeruginosa, nonhemolytic Streptococcus, and Staphylococcus aureus. All but one positive culture were polymicrobial. The amount of tissue did not correlate with the duration of stenting and the amount of granulation tissue and number of organisms decreased after stent removal. Further prospective study of the most appropriate antimicrobial therapy is needed.