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Pneumotest-Latex (Statens Seruminstitut) was evaluated for direct serogrouping of Streptococcus pneumoniae strains in clinical samples from patients with invasive disease. The technique was accurate to its level of discrimination for 62 of 67 clinical samples (92.5%). Pneumotest-Latex would be a useful alternative for direct serogrouping of pneumococci in clinical samples.
The reference method for serotyping of Streptococcus pneumoniae is the Quellung reaction (1). Pneumotest-Latex (Statens Seruminstitut, Copenhagen, Denmark) is a simple latex agglutination procedure for partial serogrouping/serotyping of S. pneumoniae strains (10, 11). Strains are classified by Pneumotest-Latex into serotypes, serogroups, or pools of serogroups. Both the Quellung reaction and Pneumotest-Latex are intended for strains isolated in pure culture. However, for many patients with invasive pneumococcal disease (IPD), culture results are negative (7, 14). Although different PCR protocols have been applied for direct serotyping of S. pneumoniae (8, 12), the technical requirements of these assays make them difficult to use in most laboratories. Direct antigen detection in clinical samples constitutes an alternative for the diagnosis of IPD (9). The utility of some latex agglutination techniques for direct antigenic detection of S. pneumoniae in normally sterile clinical samples (NSCS) is related to the capsular serotype (4). The aim of the present study was to evaluate Pneumotest-Latex for direct serogrouping of S. pneumoniae strains in NSCS. We studied 67 NSCS (positive for S. pneumoniae by culture or PCR) from IPD patients. S. pneumoniae was isolated from cultures of 47 samples (44 blood samples and 3 cerebrospinal fluid [CSF] samples). For 20 culture-negative pleural fluids, S. pneumoniae-specific PCR assays using the pneumococcal targets pneumolysin and autolysin (encoded by ply and lytA, respectively) were positive (3). Twenty-four blood samples and the 20 pleural fluids were inoculated into Bactec bottles and incubated in the Bactec 9240 system (Becton Dickinson Microbiology Systems, Cockeysville, MD). The remaining 20 blood samples were inoculated into BacT/Alert vials and incubated in the BacT/Alert system (bioMérieux, Marcy l'Etoile, France). The three CSF samples were cultured using standard methods.
The 47 S. pneumoniae isolates were serogrouped/serotyped using Pneumotest-Latex. Overnight pure cultures of S. pneumoniae in Todd-Hewitt broth were mixed with the Pneumotest-Latex suspension antiserum panels (A-I to P-T) (11). The serotype, serogroup, or pool of serogroups was assigned according to the chessboard provided by the Pneumotest-Latex manufacturer (10). Specific serotypes were confirmed by the Quellung reaction using commercial factor antisera (Statens Seruminstitut, Copenhagen, Denmark).
We adapted a classic PCR (8) to identify serotypes 1, 3, 6A/B, 14, 19A, and 19F of S. pneumoniae to a real-time PCR using the LightCycler SYBR green format followed by melting-curve analysis. Four new primers and two TaqMan probes were designed (Table (Table1)1) to detect serotypes 5 and 7F. All primers and probes were tested against the Quellung reaction for their specificity and cross-reactivity. No other serotypes were studied by PCR. The 20 culture-negative pleural fluid samples were analyzed by this method.
For direct sample agglutination serogrouping, blood culture bottles were inoculated with blood, CSF, and pleural fluid samples, 10 μl of the supernatant from each NSCS culture was mixed with 10 μl of each latex reagent, and the results were read. Serogrouping results for the isolated strains and results from PCR analyses of NSCS were considered to be reference criteria. Table Table22 shows the distribution of the results from the reference and direct agglutination methods. Twenty-one different serotypes (1, 3, 5, 6B, 7F, 8, 9N, 9V, 13, 14, 20, 10A, 11A, 12F, 15B, 15C, 19A, 22F, 23B, 24F, and 25A) were identified among the isolates (by using Pneumotest-Latex and the Quellung reaction) and in pleural fluid samples with negative culture results (by PCR). Only one isolate was identified by the serogroup only (serogroup 33). PCR assays could not differentiate the serotypes (6A/B/C) in two pleural fluid samples and could not identify any serotype (serotypes 1, 3, 5, 6A/B, 7F, 14, 19A, and 19F were excluded) in a third pleural fluid sample.
Direct latex serogrouping of strains from NSCS was accurate to its level of discrimination (serotype or serogroup) in 62 of 67 cases (92.5%). In 30 cases (44.8%), the serotype (1, 3, 5, 8, 14, or 20) and serogroup agreed and, therefore, the latex method allowed the greatest possible discrimination among strains from NSCS. In 32 cases (47.8%), direct agglutination distinguished strains only to the level of the serogroup (6, 7, 9, 10, 11, 12, 15, 19, 22, 23, or 33). Although there was not disagreement between serogroups determined by the reference and latex methods, the latex test did not allow the maximum discrimination (to the serotype level) in these cases. PCR serotyping was optimized to detect the most common serotypes of S. pneumoniae isolated from our environment. However, in one case (1.5%), the serotype was not detectable by the PCR assays, and although the serogroup identified by direct latex agglutination (serogroup 23) agreed with the PCR result (not serotype 1, 3, 5, 6A/B, 7F, 14, 19A, or 19F), the result was considered incorrect. In four cases (6.0%), direct latex agglutination assigned the strains to pools of specific serogroups (13 and 28; 24, 31, and 40; and 25, 38, 43, 44, 45, 46, and 48). Even though serotypes identified for isolated strains (serotypes 13, 24F, and 25A) were consistent with the serogroups pooled by direct latex agglutination, these results were considered incorrect.
The Quellung reaction is time-consuming and requires experience (10). Therefore, development of molecular serotyping technology is crucial for the surveillance of IPD (7, 12). However, molecular serotyping methods are not yet widely available (14). Serogrouping of S. pneumoniae strains by agglutination assays is considered cost-effective (6). If performed directly on NSCS, it may improve the diagnosis of IPD (2). According to the level of discrimination, direct agglutination serogrouping of strains in NSCS by Pneumotest-Latex agreed with the results from the reference methods (strain and PCR typing) for most of the serogroups tested. The capsular reaction has been applied previously for simultaneous detection and typing of pneumococci in enrichment broth inoculated with nasopharyngeal swabs (5), and the direct use of Pneumotest-Latex is applicable to serotype identification for colonized carriers (13). In the present study, groups of NSCS considered to be incorrectly identified by Pneumotest-Latex did not represent technical failures of agglutination. One case was not included in the PCR analysis, and strains in four samples could not be discriminated by the latex method. Although the number of samples was low and serotype representation was incomplete, the results obtained in this study suggest that Pneumotest-Latex is a simple alternative for direct serogrouping of S. pneumoniae strains in NSCS. It provides preliminary results and could be used for screening before PCR serotyping when culture results are negative.
We thank the clinical microbiological laboratories of the Autonomous Community of Madrid, Spain, for submitting invasive isolates to the Streptococcus pneumoniae Serotypes Surveillance System. We thank Marisa Fernández, Nieves Herránz, and Nazaret Méndez for their excellent technical assistance and Carmen Betriu for her careful reading of the manuscript. We are also grateful to the Subdirección General de Promoción de la Salud y Prevención (General Subdirectorate for the Promotion of Health and Prevention) of the Autonomous Community of Madrid, Spain, for providing research facilities.
This work was partially supported by an unrestricted grant from Wyeth Farma S.A., Madrid, Spain. We declare no conflicts of interest.
Published ahead of print on 9 December 2009.