Random amplified polymorphic DNA assay as a rapid tool in screening for Neisseria meningitidis serogroup C isolates of electrophoretic type 24.

Neisseria meningitidis serogroup C (NMSC) isolates of electrophoretic type 24 (ET-24), as identified by multilocus enzyme electrophoresis, are the main cause of serogroup C meningococcal disease outbreaks and sporadic meningococcal disease in the United States. We evaluated a random amplified polymorphic DNA assay as a rapid tool to screen for isolates of ET-24 by testing 199 NMSC isolates of 51 different ETs. A sensitivity of 88% and a specificity of 87% was achieved in identification of ET-24 isolates when the patterns obtained by two primers, P1 and P5, were analyzed together.

Evaluation of pulsed-field gel electrophoresis in epidemiological investigations of meningococcal disease outbreaks caused by Neisseria meningitidis serogroup C.

Since 1990, the frequency of Neisseria meningitidis serogroup C (NMSC) outbreaks in the United States has increased. Based on multilocus enzyme electrophoresis (MEE), the current molecular subtyping standard, most of the NMSC outbreaks have been caused by isolates of several closely related electrophoretic types (ETs) within the ET-37 complex. We chose 66 isolates from four well-described NMSC outbreaks that occurred in the United States from 1993 to 1995 to evaluate the potential of pulsed-field gel electrophoresis (PFGE) to identify outbreak-related isolates specific for each of the four outbreaks and to differentiate between them and 50 sporadic isolates collected during the outbreak investigations or through active laboratory-based surveillance from 1989 to 1996. We tested all isolates collected during the outbreak investigations by four other molecular subtyping methods: MEE, ribotyping (ClaI), random amplified polymorphic DNA assay (two primers), and serotyping and serosubtyping. Among the 116 isolates, we observed 11 clusters of 39 NheI PFGE patterns. Excellent correlation between the PFGE and the epidemiological data was observed, with an overall sensitivity of 85% and specificity of 71% at the 95% pattern relatedness breakpoint using either 1.5 or 1.0% tolerance. For all four analyzed outbreaks, PFGE would have given public health officials additional support in declaring an outbreak and making appropriate public health decisions.

Diversity and prevalence of PorA types in Neisseria meningitidis serogroup B in the United States, 1992-1998.

Two hundred eighty-one sporadic Neisseria meningitidis serogroup B isolates, collected through active laboratory-based surveillance, were selected to be analyzed by PorA variable region (VR) typing to determine the prevalence of PorA types in the United States. A substantial number of distinct VR types were identified, 31 in VR1 and 41 in VR2. A total of 73 different PorA types were found, and 76. 7% of these types comprise nonprototype sequences in VR1, VR2, or both. The most prevalent PorA types were P1.7,16-20 (previously P1.7, 16i), P1.22,14, P1.22-1,14 (previously P1.22a,14), P1.7,16, P1.7-1,1 (previously P1.7d,1), P1.19,15, and P1.17,16-3 (previously P1.B,16d). No correlation was observed between the PorA types and geographic origin of the isolates. These data may aid in the design of an efficacious outer membrane protein-based vaccine by identifying the most appropriate PorA types for vaccine formulation. Studies are needed to fully evaluate the extent of cross-protection in humans among the variants and prototypes in each PorA VR family.

Molecular epidemiology of diphtheria.

Molecular subtyping of Corynebacterium diphtheriae identified significant genetic diversity within the species and led to the identification of a unique clonal group that emerged in Russia in 1990 at the beginning of the current epidemic. Strains of this group belong to a distinct electrophoretic type complex and are of ribotypes D1 and D4. Identification of the group allowed for precise monitoring of the epidemic's progression and for rapid detection of cases imported to other countries. The evolution of this clonal group was monitored, and changes were identified. Molecular analysis revealed that no amino acid substitutions have occurred in the diphtheria toxin gene of the epidemic clone strains, reaffirming the use of the current vaccine as the single most effective preventive measure. Application of molecular subtyping methods and continuous monitoring of the spread of these clones has made it possible to distinguish rapidly between epidemic, endemic, and imported cases, allowing for implementation of timely and adequate preventive measures and providing reassurance that no secondary spread resulted from importations.

Cleavase fragment length polymorphism analysis of Neisseria meningitidis basic metabolic genes.

Cleavase fragment length polymorphism (CFLP) is a subtyping system based on the property of the enzyme cleavase to recognize junctions between single- and double-stranded regions of DNA formed after denaturation and cooling. To assess the capacity of CFLP for discriminating Neisseria meningitidis serogroup B strains belonging to the electrophoretic type (ET) 5 (ET-5) complex from other serogroup B strains, 30 serogroup B N. meningitidis isolates were subtyped by CFLP with internal fragments of five housekeeping genes, adk, aspC, carA, dhp, and glnA. Two genes (glnA and carA) which demonstrated a high degree of diversity for the serogroup B isolates were then used to further evaluate the suitability of CFLP for screening 50 serogroup C N. meningitidis outbreak-associated and sporadic-case isolates with a single metabolic gene. The results were compared to those from multilocus enzyme electrophoresis (MEE), the current standard subtyping method. CFLP was able to distinguish the ET-5 complex isolates from other serogroup B isolates as efficiently as MEE. Furthermore, CFLP analysis of a single gene was sufficient to identify and cluster the serogroup C isolates belonging to the ET-37 complex from other, unrelated serogroup C isolates but was not capable of differentiating between the isolates of the major individual ETs of this complex (ET-17 and ET-24) causing most serogroup C meningococcal disease outbreaks in the United States. CFLP based on a single gene with a high degree of diversity but not under selective pressure can be applied to the rapid screening of a large number of isolates related to the recognized epidemic complex ET-5 or ET-37. Additionally, CFLP can be used as an initial screening tool to survey the amount of diversity in genes that might be used to develop a DNA sequence-based subtyping system.

Disco fever: epidemic meningococcal disease in northeastern Argentina associated with disco patronage.

Neisseria meningitidis is a leading cause of adult meningitis worldwide. From 5 to 14 August 1996, 8 cases of meningococcal disease occurred in Corrientes city (population 306,000) in northeastern Argentina. Those infected ranged in age from 15 to 45 years (median, 18.5). To determine risk factors for infection, a case-control study was done. Infecting isolates were serogrouped and underwent phenotyping by multilocus enzyme electrophoresis (MLEE) and pulsed-field gel electrophoresis (PFGE). Those infected were significantly more likely than those not infected to have had exposure to passive or active cigarette smoke or to have attended a particular disco. Isolates available from 6 case-patients were all serogroup C; all had identical MLEE and PFGE patterns. These data suggest that dance clubs or discos may be a focus of transmission of N. meningitidis among young people.

School-based clusters of meningococcal disease in the United States. Descriptive epidemiology and a case-control analysis.

OBJECTIVE: To evaluate the epidemiologic features and risk factors for multiple cases of meningococcal disease in schools. DESIGN: Population-based prospective evaluation and case-control study of clusters of meningococcal disease that occurred in schools from January 1989 to June 1994. SETTING: Surveillance conducted through state health departments in the United States. MAIN OUTCOME MEASURES: Descriptive epidemiology of school-based clusters of meningococcal disease and determinants of their occurrence. RESULTS: We identified 22 clusters of meningococcal disease in 15 states. The estimated incidence of secondary meningococcal disease among schoolchildren aged 5 to 18 years was 2.5 per 100000 population, a relative risk of 2.3 (95% confidence interval [CI], 1.6-3.3). The median number of students per cluster was 2 (range, 2-4). Of 30 subsequent cases, 10 (33%) occurred 2 or fewer days after the index case, and 22 (73%) occurred 14 or fewer days after the index case. Among the 8 schools with 2 or more cases, 50% of the additional cases occurred 2 or more days after the second case. Secondary schools (grades 7 through 12) accounted for 15 (75%) of 20 cluster schools compared with 9 (45%) of 20 matched control schools (P<.05). In 16 (73%) of 22 clusters, interaction between case patients was noted. The index patient in cluster schools was more likely than the controls to have participated in a school-based group activity 14 or fewer days before illness (matched odds ratio, 7.0; 95% CI, 0.9-57). CONCLUSIONS: Three quarters of the school clusters occurred in secondary schools, with over 70% of subsequent cases occurring within 2 weeks of the index case. Rapid initiation of a chemoprophylaxis program after 2 cases of meningococcal disease in a school would have potentially prevented 50% of subsequent cases in the clusters described.

Evaluation of the use of mass chemoprophylaxis during a school outbreak of enzyme type 5 serogroup B meningococcal disease.

BACKGROUND: A vaccine for prevention of serogroup B meningococcal disease is not available in the United States, and indications for the use of mass chemoprophylaxis for control of meningococcal outbreaks are not well-defined. In response to an outbreak of six cases of enzyme type 5 serogroup B meningococcal disease among students at a middle school, we implemented a program of mass rifampin prophylaxis and evaluated the effectiveness of this preventive measure. METHODS: Oropharyngeal cultures were obtained from 351 of the 900 students before prophylaxis; 196 participants were recultured 3 weeks later. Meningococcal isolates were subtyped and tested for rifampin susceptibility, and risk factors for disease or carriage among students were evaluated. RESULTS: No cases occurred after prophylaxis. Before prophylaxis 10% (34 of 351) of students were meningococcal carriers and 3.4% (12 of 351) carried the epidemic strain. After prophylaxis 2.5% (5 of 196) were carriers and 1.0% (2 of 196) carried the epidemic strain. Rifampin was 85% effective in eradicating carriage, and the rate of acquisition of carriage during the 3-week period was low (0.5%). Carriage persisted after prophylaxis in 4 students; 3 of these postprophylaxis isolates were rifampin-resistant. Rifampin resistance thus developed in 12% (3 of 26) of preprophylaxis isolates. Disease/epidemic strain carriage was associated with enrollment in the school band and certain other classes. CONCLUSIONS: These findings suggests that mass chemoprophylaxis may be effective and should be considered for control of school serogroup B meningococcal outbreaks. This approach is less likely to be effective for control of outbreaks affecting larger, less well-defined populations and is associated with the rapid development of antibiotic resistance.

Molecular epidemiology of diphtheria in Russia, 1985-1994.

The largest diphtheria outbreak in the developed world since the 1960s began in the Russian federation in 1990. One hundred fifty-six Corynebacterium diphtheriae strains from throughout Russia, selected for temporal and geographic diversity, were assayed by ribotyping and multilocus enzyme electrophoresis (MEE). These tests showed significant genetic diversity within the C. diphtheriae species, and ribotyping and MEE data generally correlated well with epidemiologic data. A distinct clonal group of C. diphtheriae isolates (ET 8 complex) emerged in Russia in 1990 as the current outbreak began, and as the outbreak has progressed, these organisms have made up increasingly larger proportions of the strains that are isolated. Furthermore, the main characteristic of the epidemic strains is a specific combination of ET 8 and ribotypes G1 and G4. This study confirms the epidemiologic utility of the molecular subtyping methods that detected the epidemic clone and addresses the clone's origin and relation to C. diphtheriae from throughout Russia.

Meningococcal disease in Los Angeles County, California, and among men in the county jails.

BACKGROUND: From January through March 1993, there were 54 cases of meningococcal disease in Los Angeles County, California, of which 9 occurred among men incarcerated in the county's jail system, which was 40 percent above capacity at the time. Several of the 45 patients from the community had had contact with men recently released from a county jail. METHODS: We interviewed patients from the community (n=42) and neighborhood controls matched with the patients for age, race, and ethnic group (n=84) about potential exposures. We collected and cultured pharyngeal swabs for Neisseria meningitidis from men entering the central jail (n=162), men leaving the central jail (n=379), members of the jail staff (n=121), and patients at a community health center (n=214). Meningococcal isolates were identified by serotyping and multilocus enzyme electrophoresis. RESULTS: The presence of community-acquired meningococcal disease was strongly associated with exposure to a person who had been in or worked at one of the county jails (multivariate matched odds ratio, 18.5; 95 percent confidence interval, 3.8 to 90.8; P<0.001). Pharyngeal carriage of meningococcus was significantly more frequent among men released from jail (19 percent) or entering jail (17 percent) than among workers at the jails (3 percent) or community residents seen at the clinic (1 percent). Among men entering jail, those who had previously been incarcerated were more often carriers than those who had not (21 percent vs. 7 percent, P=0.03). Of the isolates from nine community residents with serogroup C meningococcal disease, eight were the same strain as that isolated from the eight inmates with serogroup C disease. CONCLUSIONS: In this outbreak of meningococcal disease in Los Angeles County, nearly half of community residents with the disease had contact with persons who had been in a county jail. The high rates of carriage among recidivists and released inmates suggests that the men became meningococcal carriers while in jail.

Molecular subtyping of Neisseria meningitidis serogroup B: comparison of five methods.

In order to compare methods for subtyping Neisseria meningitidis serogroup B isolates, 96 isolates obtained from various locations in the United States and northwestern Europe were subtyped by five methods: monoclonal antibody (MAb)-based serotyping and serosubtyping, DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE), multilocus enzyme electrophoresis (MEE), ribotyping, and PCR-restriction fragment length polymorphism of the internally transcribed spacer region of the rRNA operon (ITS PCR-RFLP). All N. meningitidis serogroup B isolates were typeable by PFGE, MEE, ribotyping, and ITS PCR-RFLP. Only 44.8% of the isolates were completely typeable (both serotype and serosubtype determination) by MAb-based serotyping and serosubtyping. 60.4% of the isolates could be serotyped but not serosubtyped, and 90.6% of the isolates could be either serotyped or serosubtyped. Simpson's discrimination indices of diversity for the methods were as follows: PFGE, 99.7%; MEE, 99.4%; ribotyping, 98.8%; MAb serotyping, 75.8%; MAb serotyping and/or serosubtyping 97.5%; and ITS PCR-RFLP, 84.2%. The high degree of diversity observed by PFGE, MEE, and ribotyping can be explained by the fact that isolates were collected from different geographic locations at various times. PFGE, MEE, and ribotyping showed greater discriminatory abilities than MAb-based serotyping and serosubtyping or ITS PCR-RFLP.

Outbreak of serogroup C meningococcal disease associated with campus bar patronage.

Between February 1991 and April 1992, eight undergraduates at a US residential university and one at a nearby 2-year college contracted serogroup C meningococcal disease. A case-control investigation with 20 controls per case, oropharyngeal carriage surveys, and multilocus enzyme electrophoresis (MEE) of serogroup C isolates were used to identify factors contributing to the outbreak. All eight sterile-site isolates from cases were closely related by MEE and were similar (though not identical) to the strain associated with the 1991-1992 epidemic of meningococcal disease in eastern Canada. Disease was associated with cigarette smoking (p = 0.012), recent patronage of campus-area bars (p = 0.034), estimated amount of time spent in campus-area bars (p = 0.0003), and, especially, recent patronage of one specific bar, bar A (p = 0.0006; odds ratio = 23.1, 95% confidence interval 3.0-571.5). In carriage surveys, 1,528 throat cultures taken from (primarily student) noncases yielded only five (0.3%) strains that were identical by MEE to those from cases. Two of these were found among 22 cultures obtained from bar A employees in spring 1992. Some cases in this outbreak may have followed transmission of the epidemic strain in bar A. Campus bar environments may facilitate the spread of meningococcal disease among teenagers and young adults.

Meningococcal carriage, alcohol consumption, and campus bar patronage in a serogroup C meningococcal disease outbreak.

Community outbreaks of serogroup C invasive meningococcal disease are increasing in North America (L. H. Harrison, JAMA 273:419-421, 1995; L. A. Jackson, A. Schuchat, M. W. Reeves, and J. D. Wenger, JAMA 273:382-389, 1995; C. M. Whalen, J. C. Hockin, A. Ryan, and F. Ashton, JAMA 273:390-394). In a recent 15-month university outbreak, disease was linked to patronage of a specific campus-area bar, suggesting that aspects of a campus bar environment might promote meningococcal transmission (P. B. Imrey, L. A. Jackson, P. H. Ludwinski, et al., Am. J. Epidemiol., in press). To investigate this hypothesis, oropharyngeal carriage results from samples taken from 867 university health service clients and 85 campus-area bar employees during the last 3 months of the outbreak were analyzed to determine factors correlated with carriage of any strain of Neisseria meningitidis. Results were validated with data from samples from 344 health center clients and 211 campus bar employees taken 8 months after the last outbreak case. Recent alcohol consumption (adjusted prevalence odds ratio = 3.8 for > 15 versus 0 drinks in last week [P = 0.0012]) and campus bar patronage (adjusted odds ratio = 1.9 for any versus no patronage in last 2 weeks [P = 0.0122]) showed separate effects in both univariate and multiple logistic regression analyses of data from the 1992 health center clients. Prevalence of meningococcal carriage among 1992 campus bar workers was 3.8 times that among health center clients; this prevalence ratio was roughly 2.5 after adjustment for alcohol consumption and bar patronage. Recent antibiotic usage was protective (prevalence odds ratio = 0.3) among health center clients and bar workers. These findings were generally supported by the validation samples. If alcohol consumption and other aspects of the campus bar environment facilitate transmission of and/or colonization by N. meningitidis, then the introduction of a highly pathogenic substrain into the campus bar environment may provide an unusual opportunity for invasive meningococcal disease within a campus community.

Relapsing invasive group B streptococcal infection in adults.

OBJECTIVE: To study recurrent group B streptococcal infection in adults. DESIGN: Patients with more than one reported group B streptococcal infection were identified through active surveillance for this infection. Sterile-site group B streptococcal isolates were evaluated for serotype and molecular subtyping using restriction endonuclease analysis of chromosomal DNA (REAC). SETTING: All acute-care hospitals in Maryland. PATIENTS: Nonpregnant residents of Maryland 18 years of age or older. RESULTS: 22 adults had at least two group B streptococcal episodes that were separated by 2 to 95 weeks (mean, 24 weeks). Of 395 patients with invasive group B streptococcal infection who survived the first episode and were followed for at least 1 year, 17 (4.3% [95% CI, 2.6% to 6.9%]) had more than one episode. Several patients were found to have endocarditis or osteomyelitis during the second episode. Group B streptococcal isolates from both episodes were obtained from 18 of 22 patients. Of the 18 isolate pairs, 13 (72% [CI, 46% to 90%]) had identical REAC patterns; the probability that at least 13 matches would be found by chance alone was less than 0.000001. Among patients with recurrent infection caused by the same strain, the interval between episodes was shorter (mean, 14 weeks) than that among patients with recurrent infection caused by another strain (mean, 43 weeks; P = 0.05). CONCLUSIONS: Recurrent group B streptococcal infection is common among adults and in most cases appears to be caused by relapse. The optimal management of adults with a first episode of group B streptococcal infection needs to be further defined to minimize the likelihood of recurrent disease.

Listeria monocytogenes infection and serotype distribution among HIV-infected persons in Los Angeles County, 1985-1992.

Persons infected with human immunodeficiency virus (HIV) are at greater risk of infection with Listeria monocytogenes (LM) than the general population. We quantify the risk of listeriosis in persons with acquired immune deficiency syndrome (AIDS) and HIV infection in Los Angeles County (LAC) and report the LM serotype distribution among HIV-infected patients with listeriosis. Active surveillance for listeriosis was performed in LAC during most of the period from 1985 through 1992. Thirty-four (10%) of 351 nonperinatal cases of listeriosis reported in LAC from 1985 through 1992 were in HIV-infected persons, 25 of whom met the 1987 AIDS case definition. The incidence of listeriosis was 95.8 and 8.8 cases per 100,000 person-years among persons with AIDS and all HIV-infected persons, respectively, but only 1.0 case per 100,000 person-years in the total population. Excluding cases from a 1985 listeriosis epidemic associated with consumption of contaminated Mexican-style cheese, 11 (65%) of 17 HIV-infected persons with available isolates were infected with LM serotype 1/2b, whereas only 64 (31%) of 208 other persons with listeriosis and available isolates were infected with LM serotype 1/2b (odds ratio = 4.1; 95% confidence interval = 1.3-14.1). LM serogroup 1/2b may have been more common among HIV-infected persons in LAC than among other persons with listeriosis because of differences in diet or sexual practices, or to chance alone. Persons with HIV-infection, especially those with AIDS, should be educated in avoiding foods at high risk of listerial contamination, such as soft cheeses, foods sold from delicatessen counters, and undercooked chicken.

Serogroup C meningococcal outbreaks in the United States. An emerging threat.

OBJECTIVE: Multiple outbreaks of serogroup C Neisseria meningitidis have recently been reported from diverse areas of the United States. To better define the characteristics of this increasingly important problem, we reviewed data on all known serogroup C outbreaks in the United States from January 1980 through June 1993. DATA SOURCES: MEDLINE searches, Centers for Disease Control and Prevention records, state health department officials, infectious disease experts, and the meningococcal vaccine manufacturer. DEFINITION OF AN OUTBREAK: Three or more cases of serogroup C meningococcal disease within a 3-month period, either among members of a community or persons attending a single school or other institution, for which those cases represented an attack rate of at least five per 100,000 population. RESULTS: Twenty-one outbreaks of serogroup C meningococcal disease were identified; eight occurred since 1991. In 1992 and the first half of 1993, approximately 180,000 doses of vaccine were administered for outbreak control, compared with approximately 34,000 doses from 1980 to 1991. Approximately 50% of community-outbreak cases were between the ages of 5 and 24 years, compared with only 19% of sporadic serogroup C cases (P < .001). Subtyping of patient isolates indicates that outbreaks are clonal; however, at least five distinct but closely related strains have caused recent outbreaks. CONCLUSIONS: Serogroup C outbreaks are occurring more frequently in the United States. The effectiveness of preventive measures depends on early recognition; therefore, physicians should promptly report all cases of suspected meningococcal disease, and the causative serogroup should be established for every case.

Comparison of ribotyping and multilocus enzyme electrophoresis for subtyping of Listeria monocytogenes isolates.

Ribotyping was compared with multilocus enzyme electrophoresis (MEE) for subtyping 305 Listeria monocytogenes isolates from clinical and nonclinical sources. For ribotyping, EcoRI-restricted genomic DNA fragments of L. monocytogenes strains were separated by agarose gel electrophoresis, and Southern blots were probed with a cloned Escherichia coli rrnB operon (plasmid pKK3535) labeled with digoxigenin. The L. monocytogenes isolates were divided into 28 distinct ribotypes, while MEE analysis divided the same isolates into 78 electrophoretic types (ETs). On the basis of their ribotype profiles, the strains were divided into two subgroups. The ribotype alpha (RT alpha) subgroup contained serotypes 1/2a, 1/2c, and 3a, and the ribotype beta (RT beta) subgroup contained serotypes 1/2b, 3b, 4b, and 4ab. This division is in complete agreement with MEE analysis, which divides the species into two subgroups (ET groups A and B), with the same serotype distribution in each subgroup. Overall, MEE was more discriminating than ribotyping. However, in several instances ribotyping discriminated between isolates within the same ET. Ribotyping was more discriminating for serotypes 1/2a, 1/2c, and 3a (Simpson's Index for Diversity [DI] = 0.81) than for serotypes 1/2b and 4b (DI = 0.76). A substantial proportion (69%) of serotype 1/2b and 4b strains clustered in five ETs and five ribotypes. These data suggest that ribotyping and MEE do not provide adequate discrimination between strains of serotypes 1/2b and 4b. Methods such as pulsed-field gel electrophoresis and random amplified polymorphic DNA analysis should be explored for further discrimination of strains of these serotypes.