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Latex agglutination or coagglutination methods have largely superseded precipitation methods for the determination of the Lancefield group of beta-hemolytic streptococci (BHS) isolated from clinical material (7). Latex agglutination kits are available for the detection of group A, B, C, D, F, and G streptococcus antigens (2, 5). In these kits, the latex is a liquid suspension. The latex in Oxoid Dryspot Streptococcal Grouping kits (Oxoid Ltd., Basingstoke, Hampshire, United Kingdom) has a novel format. It is predried onto reaction cards or strips. This study assessed the ability of the Dryspot reagents to group BHS of groups A, B, C, D, F, and G. The strains of BHS used in the study were also tested with Oxoid streptococcal grouping reagents and Streptex (Murex, Dartford, Kent, United Kingdom), which both use conventional liquid latex suspensions.

Bacterial strains. Altogether, 230 strains were used in the first investigation and 202 were used in the second investigation. A total of 49 strains from the culture collection of Basildon Hospital and the National Collection of Type Cultures (NCTC) were used in both investigations. Strains of BHS isolated and identified in the Department of Microbiology, Basildon Hospital, from clinical material submitted for examination during this study were also used in both investigations. In the first investigation, 181 clinical isolates were tested. In the second investigation, 153 clinical isolates were tested; 60 of these had also been tested in the first investigation. The strains from the culture collections consisted of groups A (three Streptococcus pyogenes strains [including NCTC 8198], two S. anginosus strains, and one S. constellatus strain), B (four S. agalactiae strains [including NCTC 8181]), C (two S. anginosus strains, one S. dysgalactiae strain [NCTC 4335], three S. equi strains [including NCTC 9682], two S. equisimilis strains, and two S. zooepidemicus strains [including NCTC 6176]), D (one S. bovis strain and four Enterococcus faecalis strains, three of which had both D and G antigens), F (seven S. constellatus strains and five Streptococcus sp. strains [including NCTC 5389]), and G (two S. anginosus strains [including NCTC 10713], three S. canis strains [including NCTC 12191], and two large-colony group G Streptococcus sp. strains), one Streptococcus sp. group L strain, and four strains of Listeria monocytogenes. The strains from the culture collection of Basildon Hospital have been previously characterized (4-6). The clinical strains were confirmed as catalase-negative, gram-positive cocci, and their Lancefield groups were determined with Oxoid latex streptococcal grouping reagents, but the results were not revealed to me until the end of the investigation.

Grouping methods. Two formats of the Oxoid Dryspot latex reagents were evaluated, the card format and the cardboard strip format. In the card format, four spots of streptococcal grouping latex had been dried onto an area of a reaction card, one area each for groups A, B, C, D, F, and G. In the strip format, four spots of latex had been dried onto a 0.5-cm² area at the end of a strip (45 by 12 mm) of stiff paper. The strips were identified according to the Lancefield group they were intended to react with, and there were separate strips for each of groups A, B, C, D, F, and G. The cards and strips were stored at room temperature in resealable foil envelopes containing a sachet of silica gel. The strips were packaged as one group per foil envelope. All other reagents were stored at 4°C and brought to room temperature prior to use.

Oxoid Dryspot card format. Strains were extracted with Oxoid extraction enzyme or Oxoid nitrous acid reagents as described below. One drop, approximately 50 µl, of extract was added to each of the test areas on the Dryspot test card provided. This was carefully mixed into the dry spots of latex in each oval area until the latex was resuspended. A separate mixing stick was used for each latex test spot.

Oxoid Dryspot strip format. Strains were extracted with Oxoid streptococcal grouping enzyme or Oxoid nitrous acid reagents as described below. One drop, approximately 50 µl, of extract was added to each of the test areas on the reaction card provided. The area containing the dried latex was brought into contact with the extract, and the strip was pushed down so that it folded at the embossed hinge. The surface of the strip was then rotated in contact with the streptococcal extract in order to rehydrate and release the latex and mix it with the extract.

Oxoid Streptococcal Grouping kits. (i) Enzyme extraction. A 0.4-ml volume of extraction enzyme was added to a polystyrene tube (75 by 10 mm), and a culture quantity equivalent to two to five large colonies was suspended in the mixture by using a 1-µl disposable loop. The mixture was incubated at 37°C for 10 min.

(ii) Nitrous acid extraction. Three drops of extraction reagent 1 and 3 drops of extraction reagent 2 were added to a polystyrene tube (75 by 10 mm), and a culture quantity equivalent to two to five large colonies was suspended in the mixture by using a 1-µl disposable loop. Three drops of extraction reagent 3 were added to the suspension, and the contents were mixed.

Streptex. (i) Enzyme extraction. A 0.4-ml volume of extraction enzyme was added to a polystyrene tube (75 by 10 mm), and a culture quantity equivalent to two to five large colonies was suspended in the mixture by using a 1-µl disposable loop. The mixture was incubated at 37°C for 10 min.

(ii) Nitrous acid extraction. One volume of extraction reagent 1 was added to the reaction tube provided, and a culture quantity sufficient to cover the blunt end of the wooden stick provided in the kit was added to the tube and mixed with the contents. The stick was left in the reaction tube for 1 min and then 1 volume of extraction reagent 2 was added to the tube.

Results and discussion. The results obtained with both of the Dryspot methods were identical to those obtained with the liquid latex methods for the equivalent strains (Table 1). The clinical strains tested in both investigations were placed in the same group in both studies, and the results for the clinical strains were identical to those obtained by the routine diagnostic laboratory. The collection strains were correctly grouped by all of the methods used. In the first investigation, 60 strains were placed in group A when enzyme extracts were tested and 59 were placed in group A when nitrous acid extracts were tested; 53 of these strains were clinical isolates. There were 54 group B (50 clinical), 25 group C (15 clinical), 28 group D (23 clinical), 19 group F (7 clinical), and 40 group G (33 clinical) strains. In the second investigation, 43 strains were identified as members of group A when enzyme extracts were tested and 42 were placed in group A when nitrous acid extracts were tested; 40 of these strains were clinical isolates, including 10 which had been tested in the first study. There were 47 group B (43 clinical, including 10 from the first study), 25 group C (15 clinical, including 10 from the first study), 25 group D (20 clinical, including 10 from the first study), 16 group F (4 clinical), and 42 group G (35 clinical, including 10 from the first study) strains.