Michael W. Reeves

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.

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

ABSTRACT Since 1990, the frequency of Neisseria meningitidisserogroup 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.

Relapsing invasive group B streptococcal infection in adults.

Invasive infection caused by group B streptococcus (Streptococcus agalactiae) is increasingly being recognized as a substantial health problem among nonpregnant adults, particularly the elderly and those with chronic illness [1, 2]. The most common clinical presentations include skin or bone infection, bacteremia without an identified source, urosepsis, pneumonia, and peritonitis [2]. Although recurrent group B streptococcal infection has been reported to occur in both infants [3-15] and adults [16-21], no population-based data are available to quantify the risk for recurrent infection. In addition, it is unknown whether recurrent disease is caused by infection with the same strain or reinfection with another strain. After we began an active surveillance project of invasive group B streptococcal infection in Maryland, we noted that some adult patients had more than one episode of group B streptococcal infection. We therefore prospectively studied the problem of recurrent infection and supplemented our study with molecular epidemiologic methods. Methods Surveillance Active surveillance for invasive Haemophilus influenzae, meningococcal, Listeria monocytogenes, and group B streptococcal infection was initiated in November 1991 as part of the Maryland Bacterial Invasive Disease Surveillance (BIDS) project. This project is a component of the multistate National Bacterial Invasive Disease Surveillance Group, which is coordinated by the Centers for Disease Control and Prevention (CDC). The surveillance case definition is the isolation of one of the above organisms from a normally sterile body fluid, such as blood or cerebrospinal fluid, from a Maryland resident of any age with clinical signs of infection. All acute-care hospitals in Maryland participate in this project, as do hospitals in Washington, D.C., at which residents of southern Maryland frequently seek medical care. Nonhospital microbiology laboratories that process blood cultures also participate. A brief case report form is completed for each eligible case, and the bacterial isolates are submitted for species confirmation and further testing. Biweekly telephone calls are made to hospital infection control practitioners to ascertain cases not reported spontaneously. To identify unreported cases, annual on-site audits of microbiology laboratories are done by reviewing the laboratory records. Identification of Patients with Recurrent Group B Streptococcal Infection At the initiation of the surveillance project in November 1991, a computer program was developed to identify all patients with group B streptococcal infection who, on the basis of their last name and date of birth, had duplicate case report forms in the surveillance database. After noting that six adult patients had more than one admission for invasive group B streptococcal infection, we decided to prospectively study recurrent disease in all age groups, including infants. Recurrent infection was defined as two separate hospital admissions during which group B streptococcus was isolated from a normally sterile body fluid. We also included nonhospitalized patients who had an acute illness during which group B streptococcus was isolated from a normally sterile body fluid and who had a second episode. Children and adults of any age with a first episode of group B streptococcal infection after 1 November 1991 were included. To calculate the proportion of all patients with group B streptococcal infection who had more than one episode, we included only patients whose first episodes occurred by 30 September 1993; this criterion allowed a minimum follow-up of 1 year. To identify patients not detected by last name and date of birth, we also searched for duplicate street addresses or medical record numbers and matches based on the first and last name or date of birth and ZIP code. Charts were reviewed to verify that each case report form represented a distinct episode of group B streptococcal infection and to obtain clinical information. Group B streptococcal isolates received from the microbiology laboratories were confirmed for species and were serotyped. Because clinical group B streptococcal isolates are restricted to a few serotypes [22], additional subtyping methods were needed. We chose restriction endonuclease analysis of chromosomal DNA (REAC) because it has been shown to provide discriminatory power among group B streptococcal isolates of the same serotype [23]. Although multilocus enzyme electrophoresis has been shown to be of limited utility in subtyping group B streptococcal isolates obtained from infants [24, 25], we wished to evaluate the method among isolates obtained from adults. Selection of Control Isolates Fifty-three group B streptococcal BIDS isolates were selected as controls for REAC analysis to provide information on the genetic heterogeneity of group B streptococci in Maryland [26]. These isolates were obtained from sterile sites in nonpregnant adult residents of Maryland who had invasive group B streptococcal infection during the same time as the patients with recurrent infection. Laboratory Assays Serotyping was done by the Lancefield capillary precipitin method [27]; we used antisera to polysaccharide antigens Ia, Ib, II, III, and V and protein antigen c prepared at the CDC. Group B streptococcal isolates were further characterized by REAC using the restriction enzyme HhAI (Gibco-BRL, Gaithersburg, Maryland). This technique was done using previously published methods [23], although 0.7% rather than 1% gels were used to improve the resolution of the larger bands. Although REAC results are somewhat cumbersome to read because of the many bands, we chose REAC as the main subtyping method because it is relatively easy to do and because it provides greater discrimination among group B streptococcal isolates than does ribotyping [23]. Because of the difficulties in interpreting subtle differences in REAC patterns, strains with similar REAC subtypes were rerun side by side on the same gel. Group B streptococcal strain extracts were prepared, and multilocus enzyme electrophoresis analysis was done in 11.5% starch at a pH of 8.0 as previously described [28]. Gel slices were stained for the following enzymes: alcohol dehydrogenase, lactate dehydrogenase, nicotinamide-adenine dinucleotide (NAD)-dependent glyceraldehyde 3-phosphate dehydrogenase. NAD phosphate (NADP)-dependent glyceraldehyde 3-phosphate dehydrogenase, NADP-dependent glutamate dehydrogenase, reduced form of NADP diaphorase, indophenol oxidase, nucleoside phosphorylase, aspartate aminotransferase, hexokinase, carbamylate kinase, phosphoglucomutase, esterase, leucine aminopeptidase, arginine aminopeptidase, leu-gly-gly peptidase, phe-leu peptidase, aldolase, and phosphoglucose isomerase [29]. Electrophoretic variants of each enzyme activity were considered to be different alleles of that enzyme and were assigned separate allele numbers. Each strain was characterized by a list of allele numbers for the different enzymes, and each unique list of alleles was designated as an electrophoretic type and assigned a separate electrophoretic type number. Statistical Analysis The number of nonpregnant persons 18 years of age or older was estimated using 1990 census-based estimates of the 1 January 1993 population minus the total number of live births among Maryland residents in 1991, the last year for which complete birth data were available. We used the Kruskal-Wallis test to analyze continuous variables and used standard methods to calculate 95% CIs [30]. The probability of at least the number of observed intrapatient REAC subtyping matches was calculated using an independence assumption model in which each permutation of the second episode results was equally probable. We did the calculations by simulation, conditioning on the results of the first episode, and randomly permuting the results of the second episode 700 000 times. With no matches, this yields P values of less than 0.00001 with a probability of 0.999. Results Seven hundred fifty-one patients with invasive group B streptococcal infection were reported between 1 November 1991 and 30 September 1993; 449 (60%) of these were nonpregnant adults 18 years of age or older. The annual incidence of invasive group B streptococcal infection among nonpregnant Maryland residents during the study period was 6.7 per 100 000 persons, 13 times the incidence of adult invasive meningococcal disease during the same time period. Of the 449 adult patients, 54 (12%) were known to have died during the first identified episode by the time the case report forms were completed. The median duration of follow-up among the 395 survivors was 23 months (range, 12 to 35 months). Among the survivors, 17 (4.3% [95% CI, 2.6% to 6.9%]) had a second episode of invasive group B streptococcal infection. Among the 17 second episodes, 1 occurred during the last 2 months of 1991, 5 occurred during 1992, 6 occurred during 1993, and 5 occurred during the first 9 months of 1994. The patients resided in 17 ZIP code regions and in 8 of the 24 Maryland administrative jurisdictions. An additional 5 patients with recurrent infection were identified with first-episode onset dates after 30 September 1993; thus, a total of 22 adult patients with recurrent infection were identified (Table 1). For the first episodes, group B streptococcus was isolated from blood in 20 patients and from the synovial fluid of the knee in 2 patients (patients 4 and 11). Group B streptococcus was isolated from blood for 21 of the second episodes and from the synovial fluid for 1 (patient 11). Patients received at least one antibiotic agent to which their group B streptococcal isolates were susceptible. The second episode occurred an average of 24 weeks after the first episode (median, 10 weeks; range, 2 to 95 weeks). Table 1. Clinical and Group B Streptococcal Isolate Analysis for Adults with Recurrent Infection* Table 1Continued Only one patient (pa

Sequence Diversity of Neisseria meningitidis 16S rRNA Genes and Use of 16S rRNA Gene Sequencing as a Molecular Subtyping Tool

ABSTRACT We investigated the diversity of the primary sequences of 16S rRNA genes among Neisseria meningitidis strains (Men) and evaluated the use of this approach as a molecular subtyping tool. We aligned and compared a 1,417-bp fragment of the 16S rRNA gene from 264 Men strains of serogroups A, B, C, and Y (MenA, MenB, MenC, and MenY, respectively) isolated throughout the world over a 30-year period. Thirty-one positions of difference were found among 49 16S types: differences between types ranged from 1 to 14 positions (0.07 to 0.95%). 16S types and serogroups were highly associated; only 3 out 49 16S types were shared by two or more serogroups. We have identified 16S types that are exclusively associated with strains of certain hypervirulent clones: 16S type 5 with MenA subgroup III, 16S type 4 with the MenB electrophoretic type 5 (ET-5) complex, and 16S types 12 and 13 with MenC of the ET-37 complex. For MenC strains, 16S sequencing provided the highest sensitivity and specificity and the best overall association with the outbreak-related versus sporadic isolates when compared with pulsed-field gel electrophoresis, multilocus enzyme electrophoresis, and multilocus sequence typing. We demonstrated for the first time an unexpected diversity among 16S rRNA genes of Men strains, identified 16S types associated with well-defined hypervirulent clones, and showed the potential of this approach to rapidly identify virulent strains associated with outbreaks and/or an increased incidence of sporadic disease.

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 epidemics 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.

Restriction fragment length polymorphism in four virulence-associated genes of Listeria monocytogenes

We observed restriction fragment length polymorphism in 4 genes of Listeria monocytogenes associated with virulence. Using the polymerase chain reaction (PCR) and primers derived from published sequences, we amplified the following genes: hlyA coding for listeriolysin O, iap coding for a putative invasion-associated factor, mpl coding for a metalloprotease, and the prfA gene that positively regulates the hylA gene. PCR-amplified DNA were cut with several restriction endonucleases, and the restriction profiles from 29 strains, representing 6 serovars (serovars 1/2a, 1/2b, 1/2c, 3a, 3b and 4b) were compared. Based on these restriction profiles, the strains were categorized into 2 subgroups: one group contained all 10 strains of serovars 1/2a, 1/2c and 3a, the other group contained all 19 strains of serovars 1/2b, 3b and 4b. This division is in complete agreement with multilocus enzyme electrophoretic analysis data which divide the species into the same 2 subgroups. Whether the differences observed in the nucleotide sequences of the 4 virulence-associated genes for the 2 subgroups of L. monocytogenes represent salient variations in pathogenic mechanisms is not known.

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 countys 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.

Ribosomal DNA fingerprinting of Listeria monocytogenes using a digoxigenin-labeled DNA probe.

A nonisotopic ribosomal DNA fingerprinting technique was developed for the characterization of Listeria monocytogenes. Plasmid pKK3535 (a pBR322-derived plasmid containing rrnB ribosomal RNA operon of Escherichia coli) was labeled with digoxigenin-II-dUTP by random priming and used to probe EcoRI fragments of L. monocytogenes chromosomal DNA on nylon filters. The method was successfully applied to the characterization of two sets of patient and food isolates of L. monocytogenes.

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.

Molecular epidemiology of sporadic (endemic) serogroup C meningococcal disease

Understanding the basis of sporadic (endemic) meningococcal disease may be critical to prevention of meningococcal epidemic outbreaks and to understanding fluctuations in incidence. Active, prospective, population-based surveillance and molecular epidemiologic techniques were used to study sporadic serogroup C meningococcal disease in a population of 2.34 million persons (Atlanta area). During 1988-1994, in which no outbreaks or case clusters were reported, 71 patients developed sporadic serogroup C meningococcal disease (annual incidence, 0.51/100,000). Eighty-three percent of patients were >2 years old. By multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, and serotyping, 84% (52/62) of the isolates available for study were identical or closely related members of the electrophoretic type 37 (ET 37) complex responsible for multiple serogroup C outbreaks in the United States in the 1990s. Sporadic disease caused by 9 clonal strains occurred over periods up to 4 years and accounted for 45% (28/62) of cases. Sporadic serogroup C meningococcal disease was most often due to a limited number of related strains that appear to slowly circulate in the population.