Phase contrast microscopy improves the visibility of the capsule, however it is not essential in conducting the Quellung reaction. Since publication of our previous recommendation, 11 European reference laboratories participated in the validation of pneumococcal serotyping

[98]. A high degree of agreement was found between the Quellung test and other serotyping methods, including latex agglutination and gel diffusion. Specifically, there was no significant difference in the percentage of mistypings (39 out of 735 serotypings) by the Quellung method (5.2%, six laboratories) compared to the non-Quellung methods (5.7%, five laboratories) [98]. An inter-laboratory quality control program conducted in four laboratories over ten years found a serotyping concordance of 95.8% Tyrosine Kinase Inhibitor Library in vitro using Quellung [99]. Although costly and time-consuming, the Quellung reaction may be preferred in laboratories with suitably experienced staff and a comprehensive set of antisera. Compared with Quellung, latex agglutination is less expensive, easier to learn, and does not require a microscope. It may therefore INCB024360 mw be more suitable for settings with limited budgets and training capacity. Commercial reagents are available; alternatively latex reagents can be produced and validated in-house. In the latter

case antibodies from commercial antisera are passively bound onto latex particles under aseptic

conditions [100] and [101]. Latex reagents produced in-house must undergo careful quality control. Reagents are stored at 4 °C. As the long-term viability of these reagents is unknown, they should be quality control tested at least annually. Reactions should be conducted using reagents at room-temperature, on a glass surface, using a consistent inoculum of fresh, low passage pneumococci. Recently, a variety of new serotyping methods have been developed including phenotypic methods that rely on antigen detection, and those that are genotype based. Several of these new methods are summarized in Table 3. Examples of genotypic methods include microarray [102], [103], [104] and [105], single or multiplex real-time PCR ([106] and [107], Rolziracetam Paranhos-Baccalà et al., unpublished data), singleplex PCR combined with sequencing [108] and [109] and multiplex PCR [110], [111] and [112]. Multiplex PCR products are usually detected by gel electrophoresis, but may also be detected by mass-spectrometry [113], DNA hybridization [114] and [115] or automated fluorescent capillary electrophoresis [116] for example. Phenotypic methods include the dot blot assay [117] and [118], latex agglutination (see Section above) and bead-based assays on a flow-cytometry or Luminex-based platform [119], [120], [121], [122], [123] and [124].