The emerging diversity of the electrophoretic types of Vibrio cholerae in the Western Hemisphere.

Since the Latin American cholera epidemic began in 1991, 447 isolates of Vibrio cholerae O1 from the Western Hemisphere have been assayed by multilocus enzyme electrophoresis (MEE) to determine allelic variation among 16 enzyme-encoding genes. Two electrophoretic types (ETs) were identified among toxigenic isolates from Latin America: 323 were ET 4, the ET associated with the Latin American epidemic, and 29 were ET 3. Twenty-three of these ET 3 isolates had a distinctive antimicrobial resistance pattern also seen in isolates imported into the United States from Latin America and Southeast Asia. These resistant isolates had an identical ribotype and nearly identical pulsed-field gel electrophoresis (PFGE) patterns. Most nontoxigenic isolates analyzed were not precursors or descendants of toxigenic epidemic strains. MEE provided a population genetic frame-work for the interpretation of PFGE and ribotype data from the isolates in this study. All three methods identified 2 distinct strains of toxigenic V. cholerae O1 currently epidemic in Latin America.

Molecular subtyping of toxigenic Vibrio cholerae O139 causing epidemic cholera in India and Bangladesh, 1992-1993.

Since October 1992, > 150,000 cases of cholera have been reported from India and Bangladesh; the great majority of Vibrio cholerae isolates belong to the newly established serogroup O139. To better understand the interaction of genetic and epidemiologic factors responsible for their sudden appearance and rapid spread, representative toxigenic V. cholerae O139 isolates were molecularly characterized and compared with a set of toxigenic V. cholerae O1 and non-O1/non-O139 strains. DNA sequences of the cholera toxin B subunit gene and multilocus enzyme electrophoresis markers of V. cholerae O139 strains were identical to those of V. cholerae O1 isolates of the seventh pandemic. Two distinct ribotypes and four pulsed-field gel electrophoretic patterns were observed for O139 strains. V. cholerae O139 strains were very similar to V. cholerae O1 strains of the seventh pandemic but clearly different from the toxigenic V. cholerae strains of serogroups other than O1 and O139.

Molecular subtyping of Vibrio cholerae O1 strains recently isolated from patient, food and environmental samples in Spain.

Nineteen Vibrio cholerae O1 strains isolated in Spain from patient, food and environmental samples in the period 1990-1992 were characterized by detection of cholera toxin by enzyme immunoassay, detection of cholera toxin gene by polymerase chain reaction, and by biotyping, ribotyping and pulsed-field gel electrophoresis. Ten isolates were toxigenic and were further characterized by multilocus enzyme electrophoresis. Molecular subtyping methods allowed precise differentiation between isolates, indicating their geographic origin. Isolates associated with the ongoing seventh pandemic were distinguishable from those associated with the present Latin American epidemic. All isolates from the environment and seafood were nontoxigenic, and were genetically different and more diverse than toxigenic isolates. The data suggest that a focus of endemic cholera does not exist in Spain, and that the analyzed nontoxigenic Vibrio cholerae O1 isolates from imported seafood were not a threat to public health.

The molecular epidemiology of cholera in Latin America.

To explain the sudden appearance and rapid spread of cholera in Latin America in January 1991, molecular techniques were used to define Vibrio cholerae O1 isolates from around the world. Restriction fragment length polymorphisms of rRNA and ctxA genes, DNA sequence of cholera toxin B subunit gene ctxB, and multilocus enzyme electrophoresis data were used to characterize 197 isolates. Worldwide, there are at least four distinct toxigenic El Tor V. cholerae O1 clones: the seventh pandemic (Eastern Hemisphere), US Gulf Coast, Australian, and Latin American. Nontoxigenic V. cholerae O1 previously isolated in Brazil, Mexico, and Peru are unlike current toxigenic isolates. The Latin American clone probably represents an extension of the seventh pandemic into the Western Hemisphere, while the US Gulf Coast clone most likely evolved separately. These data will be useful in monitoring the spread of cholera, determining the origin of outbreaks in both hemispheres, and implicating specific vehicles of transmission.

Genetic diversity among toxigenic and nontoxigenic Vibrio cholerae O1 isolated from the Western Hemisphere.

Multilocus enzyme electrophoresis was used to examine genetic relationships among and between toxigenic and non-toxigenic isolates of Vibrio cholerae O1 obtained from patients and the environment in the US Gulf Coast and surrounding areas. A total of 23 toxigenic and 23 non-toxigenic strains were examined. All the toxigenic and 7 of the non-toxigenic strains had the same alleles at 16 enzyme loci, whereas the balance of the nontoxigenic strains had 9 distinct combinations of alleles. This study suggests that all of the toxigenic strains belong to a single clone, and that while some of the non-toxigenic isolates were related, most were of diverse origin.

Evaluation of 10 methods to distinguish epidemic-associated Campylobacter strains.

We compared four phenotypic and six genotypic methods for distinguishing Campylobacter jejuni strains from animals and humans involved in four epidemics. Based on a comparison with epidemiologic data, the methods that correctly identified all strains in three milkborne outbreaks and one waterborne outbreak were heat-stable and heat-labile serotyping; multilocus enzyme electrophoresis (MEE); DNA restriction endonuclease analysis with BglII, XhoI, PvuII, or PstI; and Southern blot and hybridization of PvuII- and PstI-digested DNA with Escherichia coli 16S and 23S rRNA (ribotyping). Biotyping, phage typing, plasmid analysis, and probing of BglII and XhoI DNA digests with C. jejuni 16S rRNA genes failed to correctly separate one or more strains. MEE, restriction endonuclease analysis, and ribotyping were the most sensitive methods and identified nine types among the 22 strains. These methods were also capable of further distinguishing strains within the same serotype. Data from MEE were also analyzed to calculate genetic relatedness among strains. Serotyping was the most discriminating phenotypic method, with eight and seven types distinguished by the heat-stable and heat-labile methods, respectively. MEE and ribotyping had several advantages over the other methods because they measure relatively stable and significant chromosomal differences and are applicable to other species and genera. These methods, however, are complex and not easily quantified; they are currently limited to specialized laboratories. When antisera are available, serotyping appears to be an effective and more practical approach to the identification of epidemic-related strains.

Clonal nature of Salmonella typhi and its genetic relatedness to other salmonellae as shown by multilocus enzyme electrophoresis, and proposal of Salmonella bongori comb. nov.

Crude cell extracts of 26 isolates of Salmonella serotype typhi (S. typhi) and 48 other Salmonella isolates representing 28 serotypes and seven DNA hybridization subgroups were analyzed for electrophoretic variants of 24 metabolic enzymes by starch gel electrophoresis. All strains of S. typhi had identical isoenzyme patterns, indicating that they were a single clone. All of the enzymes detected in the remaining strains were polymorphic, and the degree of genetic variation was quite high. The average number of alleles per enzyme locus was 4.7, and the mean genetic diversity per locus was 0.556. Thirty-two distinct allele profiles, or electrophoretic types (ETs), were found in these 48 strains of Salmonella serotypes other than S. typhi. Analysis of the genetic relationships of the ETs to each other showed that, with one exception, the ETs formed subgroups that were consistent with the subgroupings based on DNA hybridization studies. ET profiles were not always linked to specific serologic patterns. These data show that multilocus enzyme electrophoresis has a potential application in epidemiologic and taxonomic studies of salmonellae, although it is not differential for S. typhi. We also propose a new species, Salmonella bongori comb. nov., a new combination base on the elevation of Salmonella choleraesuis subsp. bongori to the level of species.

Quality of commercially produced Shigella serogrouping and serotyping antisera.

Shigella grouping antisera from five manufacturers and typing antisera from two were purchased and evaluated with homologous and heterologous Shigella strains in the slide agglutination test. Only 31 of 73 (42%) antisera were satisfactory. In many instances, the antisera gave negative, as opposed to weak, reactions when they should have given strong positive reactions. Four reagents cross-reacted with Shigella strains. Of the 19 polyvalent grouping antisera to subgroups Shigella dysenteriae serotypes 1 through 7, S. flexneri serotypes 1 through 6, S. boydii serotypes 1 through 7, and S. sonnei forms I, II, only one S. sonnei reagent and five S. flexneri reagents were satisfactory with greater than or equal to 90% of the homologous strains. The reagent of poorest quality was satisfactory with only 18% of the homologous strains. There were three polyvalent antisera to the higher types of S. dysenteriae and S. boydii, which were available from only one company, that adequately identified 80, 63, and 65% of the homologous strains. Typing antisera were available from only two companies, and 30 of 51 (59%) were satisfactory. Commercially available Shigella antisera are inadequate for the laboratory testing required for planning the development of and evaluating Shigella vaccines.

Panel of reference strains for evaluating serologic reagents used to identify gonococci.

A panel of strains for evaluating Neisseria gonorrhoeae serologic reagents was developed. The strains selected for the panel were antigenically diverse and representative of strains isolated worldwide and had been isolated from a variety of anatomic sites. A few strains with characteristics that can cause problems in serologic tests were included. The panel of 52 gonococcal and 20 nongonococcal strains was used to evaluate two commercially produced kits with monoclonal antibody reagents, GonoGen and Phadebact, and one Phadebact kit with absorbed rabbit antiserum. The GonoGen reagent correctly identified all gonococcal strains and did not react with any of the nongonococcal strains. The Phadebact absorbed antiserum reagent correctly identified 47 of 48 gonococcal strains but reacted with 2 of the 20 nongonococcal strains. The Phadebact monoclonal antibody reagent correctly identified all the gonococcal strains; however, it gave positive reactions with 8 and trace reactions with 4 of the 20 nongonococcal strains.

Characterization of Neisseria gonorrhoeae reference strains used in development of serologic classification systems.

Certain strains of Neisseria gonorrhoeae have been used by numerous investigators to develop serologic classification systems. Some of these strains have been used by investigators to study gonococcal virulence. A reference consisting of strain classification by auxotype and serovar, a strain history, and a selected bibliography are provided cohesively.

Comparison of serologic screening tests for brucellosis.

The slide agglutination test (SAT), microagglutination test (MAT), and card agglutination test (CAT) were compared with each other, using the tube agglutination test (TAT) as the standard method, by two reference laboratories to determine effectiveness as screening tests for human brucellosis. TAT titers of 1,253 sera tested in both laboratories were compared. In one laboratory, 1,270 sera were tested by the TAT and SAT, while the other laboratory tested 1,261 sera by both methods. Of these sera, 1,155 were tested in one laboratory by the CAT and 187 sera were tested by the MAT. Compared with that of the TAT (greater than or equal to 160 positive), the sensitivities were 97 to 100% (SAT), 90% (CAT), and 88% (MAT). The specificities were 88 to 89% (SAT), 98% (CAT), and 88% (MAT). For populations with a low prevalence of disease, increased specificity offers higher predictive value, so the CAT and MAT are preferable for screening purposes and the choice between tests depends on the number and frequency of tests performed. All sera reactive in the CAT and MAT should be retested with the TAT.

Evaluation of API SerImm Sure strips for screening and grouping Salmonella isolates.

Two new latex agglutination products in which the reagents are dried in macrocupules in plastic strips were evaluated. API SerImm Sure Salmonella Poly for screening and API SerImm Sure Salmonella for grouping (Analytab Products, Plainview, NY) were tested with 63 recent isolates and 72 stock strains of Salmonella groups A-E and 43 stock strains of Salmonella groups F-67. Of the homologous strains, the polyvalent reagent correctly identified 89%, and the grouping reagents 90%. The most commonly occurring serotypes of groups A-E were identified. The major problems were failure to identify Group B 1,4,12,27, and group D29,46 strains, and lack of macrocupules with normal serum-latex controls. The major advantage of the products is convenience, since the SerImm Sure packaging eliminated the need to dilute anti-sera and prepare slides.

The development by the Centers for Disease Control of a specification for streptococcal serogrouping kits and its application to Streptex and to the Phadebact Streptococcus Test.

A Centers for Disease Control (CDC) specification for evaluating commercially produced streptococcal agglutination reagents was developed and used to test Streptex and Phadebact Streptococcus Test kits. Evaluation methods and performance requirements were based on product claims made in the package labelling. Except for the reagent for Streptococcus group D, reagents of both systems identified 100% of the blind-coded reference strains when follow-up methods were done according to the manufacturers' directions. Streptex group D reagent did not identify all group D strains, but the manufacturer instructed the user to test with other methods in certain circumstances. The interaction of personnel of the Center for Infectious Diseases (CID), CDC, with commercial producers and consumers in a pre-market evaluation program is described.

Assessment of availability and efficacy of commercial Salmonella grouping antisera.

Salmonella somatic antisera for groups A to E were purchased from four commercial producers directly by the Center for Disease Control (CDC) and indirectly through two hospitals. CDC specifications and methods were used to evaluate antisera shipped directly to CDC. To assess the performance of the products under simulated user conditions, we used the commercial antisera purchased indirectly through the hospitals to group coded cultures. Of the 23 antisera ordered by CDC and the hospitals, the CDC received all 23, a large medical complex received 20, and a private hospital received 9. Similar results were obtained with producer and CDC test methods. Forty-five different lots of antisera were evaluated, of which 20% did not meet CDC specifications. The CDC specifications and latest revisions are discussed.

Evaluation of commercial Salmonella O polyvalent and Vi antisera.

Salmonella somatic polyvalent antisera for groups A through E or A through I with or without Vi and Salmonella Vi antisera were purchased from four commercial producers and evaluated. Center for Disease Control (CDC) methods and specifications were used. The four polyvalent and two of the four Vi antisera did not meet CDC performance specifications. Suggestions are made for improving the laboratory usefulness of the reagents and the product labeling.