Infections with Streptococcus pneumoniae are a significant problem for young children and older adults. Even with effective antibiotic treatment, patients with pneumococcal infections can have serious sequelae, and there is a rapid spread of antibiotic-resistant S. pneumoniae. Thus, the ability to prevent pneumococcal disease with vaccines remains critical to the optimal management of the pneumococcal disease. Although the 23-valent polysaccharide (PS) vaccine is currently available, it is not immunogenic in young children and fails to protect at least 30% of the immunized elderly (10). In an effort to prepare vaccines effective among young children and more effective in the elderly, conjugate vaccines containing PS linked to a protein moiety, analogous to the Haemophilus influenzae type b vaccine, are being developed with up to 11 PS serotypes in clinical trials (9).Once the vaccine is licensed, its effectiveness will need to be monitored. Since the protection provided by the pneumococcal vaccines is serotype specific, the effective vaccine should selectively reduce the prevalence of S. pneumoniae expressing the vaccine serotypes without altering the prevalence of S. pneumoniae expressing the nonvaccine serotypes (1). In addition, S. pneumoniae has been shown to undergo in vivo transformation of capsular serotypes (8), and the vaccine may result in an increased frequency of strains with nonvaccine serotypes. The vaccine may therefore even force the appearance of new, virulent pneumococcal strains expressing some of the nonvaccine serotypes. Thus, serotypes of S. pneumoniae need to be monitored following the introduction of new pneumococcal vaccines.Serotyping S. pneumoniae is not simple because over 20 serotypes are common among clinical isolates. Although several serotyping methods are available (2, 5, 7), the present typing system is laborious and slow and requires considerable technical experience. Consequently, all these methods are ill suited for efficacy studies of new vaccines, and there is a need for new techniques that can rapidly and reliably determine pneumococcal capsular serotypes of a large number of isolates. We report the development and evaluation of a simple and efficient flow cytometric method, with which a pneumococcal isolate could be simultaneously tested for 15 of the most common serotypes. MATERIALS AND METHODSPreparation of bead set coated with different pneumococcal capsular PS serotypes. Latex beads of five different diameters ranging from 2 to 4.76 m were obtained from Bangs Laboratories (Fisher, Ind.). They were dyed with Did oil (Molecular Probes, Eugene, Oreg.) to prepare them with three different levels (none, low, and high) of red fluorescence. To dye the beads with Did oil, they were mixed with the dye in dimethyl sulfoxide and incubated overnight with shaking at room temperature. The range of dye concentration for beads was 1 to 10 g/ml for a low level of fluorescence and 1 to 5 mg/ml for a high level of fluorescence. The beads were washed with 0.25% Triton X-100 several times...

Assays measuring opsonophagocytic killing capacity of immune sera are good surrogate assays for assessing pneumococcal vaccine responses, but they are tedious to perform primarily because the enumeration of surviving bacteria requires the counting of individual bacterial colonies. To overcome this limitation, we have developed a simple and rapid chromogenic assay for estimating the number of surviving bacteria. In this method, the conventional opsonophagocytic killing assays were performed in microtiter wells with differentiated HL-60 cells as phagocytes. At the end of the assay the reaction mixture was cultured for an additional 4.5 h to increase the number of bacteria. After the short culture, XTT (3,3-[1{(phenylamino)carbonyl}-3,4-tetrazolium]-bis[4-methoxy-6-nitro] benzene sulfonic acid hydrate) and coenzyme Q were added to the wells and the optical density at 450 nm was measured. Our study shows that changes in the optical density were proportional to the number of CFU of live bacteria in the wells. Also, the number of bacteria at the end of the 4.5-h culture was found to be proportional to the original number of bacteria in the wells. When the performance of the chromogenic assay was evaluated by measuring the opsonizing titers of Streptococcus pneumoniae serotypes 6B and 19F, the sensitivity and precision of the new method were similar to those of the conventional opsonization assay employing the colony counting method. Furthermore, the results of this chromogenic assay obtained with 33 human sera correlate well with those obtained with the conventional colony counting method (R > 0.90) for the two serotypes (6B and 19F). Thus, this simple chromogenic assay would be useful in rapidly measuring the capacities of antisera to opsonize pneumococci.