Comparison of the Denka Seiken Slide Agglutination Method to the Quellung Test for Serogrouping of Streptococcus pneumoniae Isolates

Abstract

This study compared a slide agglutination test (Denka Seiken, Tokyo, Japan) to the “gold standard” quellung reaction (Pneumotest; Statens Serum Institut, Copenhagen, Denmark) for the serogrouping of pneumococci. Two hundred clinical isolates of Streptococcus pneumoniae were used for the comparison. Each assay was performed according to the manufacturer's instructions. There was an overall agreement of 95.7% between the two methods. Only 4 of 10 isolates of serogroup 22 were detected with the slide agglutination assay. Two isolates that were untypeable by the Pneumotest method were typed as serogroups 6 and 31 by the slide agglutination method. The Pneumotest method was unable to type 22 isolates, and the slide agglutination method was unable to type 16 isolates. The slide agglutination method compares favorably with the Pneumotest method and is easier to perform and to interpret.

Streptococcus pneumoniae is the major bacterial cause of acute otitis media, sinusitis, and community-acquired pneumonia. More serious invasive illnesses include bacteremia and meningitis. Mortality due to pneumococcal infections is high, especially in developing countries (6). Penicillin has been an effective treatment for these conditions; however, the growing number of penicillin-nonsusceptible strains of S. pneumoniae has become an increasing concern. In addition to the increase in numbers of penicillin-nonsusceptible strains, S. pneumoniae is now becoming increasingly resistant to other antibiotics, leaving vancomycin as the last resort for treatment (7, 9).

Pneumococci are carried in the nasopharynges of healthy individuals, especially in children, where it is the main source of person-to-person transmission. Increasing antibiotic use and development of resistant strains have led to the carriage of resistant strains of pneumococci (7).

Vaccines against the prevalent serogroups of S. pneumoniae causing invasive disease are available. The 23-valent capsular polysaccharide vaccine and the newer heptavalent polysaccharide-protein conjugate vaccine include the serogroups most often responsible for bacteremia, meningitis, pneumonia, and otitis media (1, 4, 7). Preliminary studies suggest that the pneumococcal conjugate vaccine reduces carriage of the serogroups included in the vaccine (9).

There are 90 serogroups of S. pneumoniae, based upon the antigenic differences in the capsular polysaccharide. Isolates with high-level resistance to β-lactam antibiotics often belong to prevalent, well-documented clonal groups. A majority of these clonal groups have also developed resistance to other antibiotics (9). Serogrouping of S. pneumoniae strains has therefore become increasingly important to monitor the serogroups prevalent in the general population and to track resistance patterns. It is also important to assess the efficacy of the newly developed vaccines by testing isolates recovered from infections in previously vaccinated patients (9).

The “gold standard” for serogrouping S. pneumoniae isolates has been the quellung reaction (Pneumotest; Statens Serum Institut, Copenhagen, Denmark) (5). Serogrouping by the Pneumotest reaction is time-consuming and requires a microscopic examination and skill and experience to interpret the results (2). A slide agglutination method by Denka Seiken Co. potentially offers a faster, less-demanding method for the serogrouping of pneumococci. The goal of this study was to evaluate the slide agglutination assay for pneumococcal serogrouping by testing S. pneumoniae strains that were previously characterized by the Pneumotest reaction.

The S. pneumoniae isolates used in this study were recovered from nasopharyngeal cultures from children who were at least 6 months of age and less than 6 years of age, living in isolated rural communities in Utah and Idaho. This 2002 S. pneumoniae surveillance study was conducted by the Departments of Internal Medicine, Family and Preventive Medicine, and Pathology, University of Utah, Salt Lake City, and was described in detail previously (8). The study sampled 1,484 children, producing 485 isolates that were positive for S. pneumoniae. A sample of 200 isolates, which represented all of the serogroups obtained from the surveillance study, was used for this comparison. The isolates obtained were frozen at −70°C in brain heart infusion (BHI) broth-glycerol stock solution (9.9% [vol/wt] glycerol, 3.33% [wt/vol] BHI broth; ARUP Laboratories Reagent Lab). Frozen isolates were subcultured onto Columbia agar with 5% sheep blood (BD BBL, Sparks, Md.), streaked for isolation, and incubated for 24 h at 35°C in 5% CO₂. This process was repeated twice to prepare working cultures. Isolates were identified as S. pneumoniae by colony morphology, alpha hemolysis, susceptibility to an optochin disk (BD BBL), and solubility in bile (11.1% [wt/vol] sodium deoxycholate [ARUP Laboratories Reagent Lab]).

These pneumococcal strains were serogrouped by the Pneumotest reaction by using the procedure described in the product package insert from Statens Serum Institut. The Pneumotest kit consists of 12 pooled antisera and uses a chessboard schema, which places strains of S. pneumoniae into 34 groups or types (3). The chessboard schema is shown in Table 1.

TABLE 1.

Pneumotest chessboard for identification of pneumococcal groups or types^a

Pooled serum	Serotype or group reacting with indicated serum pool					Nonvaccine types or groups reacting with pooled serum
	P	Q	R	S	T
A	1	18	4	5	2
B	19	6	3	8
C	7				20	24, 31, 40
D			9		11	16, 36, 37
E			12	10	33	21, 39
F				17	22	27, 32, 41
Gb						29, 34, 35, 42, 47
H	14	23		15		13, 28
Ib						25, 38, 43, 44, 45, 46, 48

^aThe five pooled sera P to T are composed in such a way that each of the 21 vaccine-related types and/or groups (indicated by boldface) reacts with both one of these sera and one of the seven pooled sera A to F plus H, while other nonvaccine types and groups react with one pooled serum only (A to I). Pools G and I do not react with vaccine types.

^bPools G and I are not included in the Pneumotest kit.

The anticapsular sera provided in the slide agglutination method have been placed into a serotyping schema based on the Pneumotest kit with some modifications made to the composition of the polyvalent antisera. The titers of these antisera were adjusted to allow slide agglutination with the S. pneumoniae bacterial cells. The typing schema for the slide agglutination method consists of 8 polyvalent and 39 monovalent serotype antisera. This schema is shown in Table 2.

TABLE 2.

Denka Seiken slide agglutination polyvalent and monovalent serotype schema

Polyvalent serotype	Monovalent serotypes
1	1, 2, 3, 4, 5
2	6, 8, 9, 10
3	11, 12, 14, 15, 16
4	17, 18, 21, 22
5	20, 29, 31, 33, 34, 35, 47
6	23, 25, 28, 41, 46
7	27, 32, 36, 38, 39
8	7, 19, 24, 40

After isolates had been subcultured from the frozen BHI-glycerol stock solution onto Columbia agar with 5% sheep blood, the working culture was used to perform slide agglutination, by following the recommended procedure supplied by Denka Seiken. These isolates were tested one at a time, with the investigators blinded to the original serogroup designation, first with the polyvalent antisera and then with the respective monovalent antisera included in the polyvalent serotype that showed agglutination. To check for spontaneous agglutination of the bacterial cells, phosphate-buffered saline was tested with each isolate. A glass slide, divided into partitions with a china marker, was used for testing. One drop of each of the polyvalent antisera (antisera 1, 2, 3, 4, 5, 6, 7, and 8) and 1 drop of phosphate-buffered saline were placed on a partition of the slide. A 10-μl loopful of bacterial cells was then placed into each partition and mixed with a loop, using a new sterile loop with each respective antiserum. The slide was rotated by hand for 30 s. Coarse aggregation of the bacterial cells with a clear background was considered a positive result for the antiserum showing agglutination. A homogenous emulsion of bacterial cells was interpreted as a negative result.

The results of the slide agglutination method were compared to the results of the Pneumotest method. Isolates that produced discordant results were tested a total of three times by each method. The serogroup obtained at least two of the three times was used for the analysis, and final results were recorded as matching, discordant, or unable to type.

A total of 200 S. pneumoniae isolates representing the 19 serogroups obtained from the surveillance study were tested. These included serogroups 4, 6, 7, 8, 9, 10, 11, 14, 15, 18, 19, 22, 23, 33, and 35 and pool C only (serogroups 24, 31, and 40), pool D only (serogroups 16, 36, and 37), pool E only (serogroups 21 and 39), and pool H only (serogroups 13 and 28). Serogroup 35 antiserum is absent from the Pneumotest kit, and serogroup 13 antiserum is absent from the slide agglutination kit.

The total numbers of serotypes within each serogroup tested by the two methods and the number and percentages of matching results between the two methods are summarized in Table 3. There were a total of 34 discordant results before repeat testing. Fourteen (41.2%) of these discordant results were due either to the absence of antiserum 35 in the Pneumotest kit (n = 12) or to the absence of serogroup 13 (n = 2) with the slide agglutination method. Excluding the results of isolates not contained in the assays tested, there were a total of 20 discordant results. The overall agreement before repeat testing was 89.2%. After repeat testing, there were a total of eight discordant results for an overall agreement of 95.7% (178 of 186 isolates). Two of these eight discordant results were found in serogroup 6 and pool C only (serogroups 24, 31, and 40); the slide agglutination method was able to detect one more isolate in each group than the Pneumotest kit.

TABLE 3.

Comparison of quellung (Pneumotest) and Denka Seiken slide agglutination methods^a

Quellung serogroup	No. (%) of isolates detected by:
	Pneumotest	Slide agglutination method
4	2	2 (100)
6	36	37 (100)
7	1	1 (100)
8	1	1 (100)
9	7	7 (100)
10	3	3 (100)
11	8	8 (100)
14	9	9 (100)
15	25	25 (100)
18	4	4 (100)
19	34	34 (100)
22	10	4 (40)
23	26	26 (100)
33	1	1 (100)
35	0b	12 (100)
C (24, 31, 40)	6	7 (100)
D (16, 36, 37)	1	1 (100)
E (21, 39)	2	2 (100)
H (13, 28)	2	0 (0)c
Non typeable	22	16

^aTwo hundred isolates were examined.

^bAntiserum 35 was not included in the kit.

^cAntiserum 13 was not included in the kit.

The most notable discordant result occurred with serogroup 22. The slide agglutination method was unable to detect 6 of 10 serogroup 22 isolates. This result was a failure of the polyvalent serogroup 4 antisera to adequately show agglutination for these isolates. The isolates in question showed strong agglutination in the monovalent serogroup 22 antisera. These six serogroup 22 isolates were sent to the Centers for Disease Control and Prevention for serogrouping. All six isolates were identified by the Centers for Disease Control and Prevention as serogroup 22. Subsequent testing of these six isolates at ARUP Laboratories with an improved polyvalent serogroup 4 antiserum, provided by the manufacturer, showed strong agglutination.

Another problem encountered during the study was the weak agglutination of the monovalent serogroup 10 antisera with the three serogroup 10 isolates included in the study. These were not considered discordant since any agglutination is interpreted as a positive result in the slide agglutination method.

This study is limited by the fact that only 25 of 40 serogroups of S. pneumoniae were available for study. However, in this short pilot study, the slide agglutination method appeared to offer some advantages over the Pneumotest method. More rapid results were obtained with the slide agglutination method. On average, slide agglutination resulted in serogroup identification in half the time (10 min/test) needed for the Pneumotest reaction (20 min/test). The reduction in the time needed to complete the slide agglutination test generated savings in labor costs ($8.33/isolate with the Pneumotest versus $4.20/isolate with the slide agglutination based upon a labor cost of $25/h). Reagent costs for the slide agglutination product have not been established by Denka Seiken at this time, since the kit has not yet been marketed.

Additional studies using more diverse isolates from adult as well as pediatric patients are required before the Denka Seiken slide agglutination method can be considered a reliable replacement for the Pneumotest. The data generated in this limited study are promising. The Pneumotest kit and slide agglutination produced comparable results, with greater than 95% agreement. The problems encountered with serogroups 22 and 10 need to be addressed. Further testing of additional serogroup 22 isolates with the improved polyvalent serogroup 4 antiserum is warranted to ensure resolution of this problem. However, following improvement in these two problem areas by the manufacturer and validation of the results of this study by others, slide agglutination may provide a more rapid, less expensive, and less demanding alternative to the gold standard quellung reaction of the Pneumotest.