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Am. J. Trop. Med. Hyg., 74(5), 2006, pp. 863-867
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene

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FIELD EVALUATION OF THE CHESSBOARD MODIFICATION FOR SEROTYPING OF STREPTOCOCCUS PNEUMONIAE IN A SMALL LABORATORY IN BANGLADESH

ELJA HERVA*, SIMO GRANAT, ZAKARIA MIA, JUKKA OLLGREN, LIISA PIIRAINEN, AND P. HELENA MÄKELÄ
National Public Health Institute, Oulu, Finland; National Public Health Institute, Helsinki, Finland; Gonoshasthaya Vaccine Research Laboratory, Dhaka, Bangladesh


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The chessboard modification of the quellung method for serotyping Streptococcus pneumoniae (Pneumococcus) was introduced in the Gonoshasthaya Vaccine Research Laboratory, a small laboratory in Bangladesh. We applied initial bench-side training and subsequent continuous surveillance and quality assurance as approaches for good laboratory practice. Results obtained a this laboratory on 1,101 consecutive isolates had satisfactory sensitivity (85.1–100%) and specificity (97.9–99.9%) for serotyping the 10 most common serogroups/types of pneumococci when compared with the results obtained in the Finnish reference laboratory for pneumococcal serotyping at the National Public Health Institute. We conclude that serotyping of pneumococci by the chessboard method can be introduced into a small laboratory by providing basic bacteriologic skills, adequate initial training, and continuous external support.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Streptococcus pneumoniae (pneumococcus) is one of the most common bacterial pathogens in humans. It causes severe, life-threatening infections such as pneumonia and meningitis, and is often associated with bacteremia that also occurs without these conditions, especially in children. Pneumococcus is also the leading bacterial pathogen in acute otitis media, the most common bacterial disease in children, and in paranasal sinusitis. Pneumococcus most commonly occurs as a part of the bacterial flora of the nasopharynx of healthy carriers, from where it can spread directly to other parts of the respiratory tract or via blood to normally sterile sites.1 The prevalence of pneumococcal carriage increases during the first two years of life; in developing countries, the colonization occurs at an earlier age and has a higher prevalence than in industrialized countries.2

To understand the epidemiology and pathogenesis of pneumococcal diseases, it is necessary to understand the ecology and epidemiology of pneumococcal carriage as the reservoir of the infection. Since the burden of serious pneumococcal diseases is heaviest in the developing world, such studies are particularly needed.3 Serotyping is the most important method for characterizing pneumococcal isolates from both patients with pneumococcal diseases and pneumococcal carriers in epidemiologic studies. The recent introduction of a vaccine that reduces not only pneumococcal disease but also carriage in a serotype-specific way has created a need for serotyping in rational planning and surveillance of vaccination programs.

Serotyping is usually performed in research or reference laboratories with highly qualified and experienced personnel and good material resources, but this is not always possible in developing countries. To extend the applicability of serotyping, Sørensen has described a simplified modification of the quellung method for serotyping of pneumococci.4 In this chessboard method, two sets of pooled antisera pools (A through I and P through T) are used, avoiding the need of the large panel of type- or group-specific antisera. The same set of antiserum pools has also been used to sensitize latex particles as reagents for serotyping by the latex agglutination method.5,6 To our knowledge, use of the original method of Sørensen in the field, developing countries, or elsewhere has not been published.

We used the chessboard method in a study on pneumococcal colonization carried out in Bangladesh at a small local bacteriological laboratory in which there was only limited experience in pneumococcal bacteriology and no experience in pneumococcal serotyping. In this report, we compare the serotyping results obtained locally with those obtained using the full set of antisera at the technical support laboratory in Oulu, Finland (KTL-Oulu), which is the national reference laboratory for S. pneumoniae in Finland.7 In this context, we describe and discuss our experiences in establishing the rather demanding bacteriologic methodology for a research project in a developing country.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study site. The study was conducted at the Gonoshastaya Vaccine Research Laboratory (GVRL) in Savar, Dhaka, Bangladesh as a collaborative project between a Finnish study group based at the National Public Health Institute (KTL) in Helsinki and Oulu, Finland and the Gonoshastaya Kendra (GK) Trust, a Bangladeshi non-governmental agency active in the health sector. Since its establishment in 1993, GVRL has carried out small research projects and functioned as a laboratory for clinical bacteriology with a small number of daily specimens.

Carriage study and subjects. The prospective community-based pneumococcal carriage study was carried out in a semi-rural area in Savar, Bangladesh, where the GK Health Program provides primary health care service to the villages. Newborn children and their family members were enrolled in the study after informed consent was obtained from parents. The families were first visited at two-week intervals and later at four-week intervals. At each visit, nasopharyngeal secretions were collected from the newborn children, their mothers, and other family members as present for isolation, identification, and serotyping of S. pneumoniae. The details of the carriage study have been preliminarily presented at the Third and Fourth International Symposia on Pneumococci and Pneumococcal diseases in 2002 and 2004 (Granat S, Zakaria MM, and others, unpublished data).

Training of personnel. In January–February 2000, four members of the study group from KTL in Finland visited GVRL to introduce the methods for the pneumococcal carriage study. Isolation of S. pneumoniae from nasopharyngeral secretions and identification and serotyping were demonstrated to a local microbiologist (ZM) and to three local laboratory technicians who also did the bench work under supervision during the visit. Later during the course of the study one or two members of the study group visited the laboratory on several occasions. For the collection and transport of nasopharyngeal secretions for culture, two teams of two para-medics (health workers of GK) were trained by a physician of the KTL group and later supervised by a local physician.

During the training period, standard operation procedures were prepared on 1) collection and transport of nasopharyngeal samples, 2) culture of the swabs for S. pneumoniae and storage of the isolates, 3) identification of S. pneumoniae and testing of penicillin sensitivity using oxacillin discs, 4) serotyping of S. pneumoniae using the chessboard modification of the quellung method, and 5) quality control of bacteriologic media and reagents. The importance of following the standard operating procedures was emphasized in the training of the personnel.

Isolation and identification of S. pneumoniae. The nasopharyngeal samples were collected with a calcium alginate swab by inserting it deep into the nasopharynx, rotating, and letting it saturate for a few seconds. The swab was then immediately inserted into a tube containing skim milk-tryptone-glucose-glycerol (STGG) medium that was immediately transferred into an insulated box containing cold chargers or dry ice.2 In the afternoon (within six hours), the box with the daily yield of swabs was carried to the laboratory where the swabs were vortexed, transferred into a freezer, and stored at –20°C.

For culture, the swabs were thawed at room temperature for 15–30 minutes, vortexed for 10 seconds, inoculated with a 10-µL loop onto gentamicin (5 µg/mL) sheep blood agar plates, and transferred to the freezer at –20°C within one hour. The plates were incubated in candle jars at 36°C for two days. Growth was inspected daily for alpha-hemolytic colonies suggestive of pneumococci. The colonies were counted, two colonies were picked and streaked separately on halves of a sheep blood agar plate, and an optochin disc (6 µg; AB Biodisk, Solna, Sweden) was put on each half. After incubation in candle jars overnight the result was read and an inhibition zone > 12 mm identified S. pneumoniae. If the zone was 10–12, mm the test was repeated, and a zone < 10 mm excluded S. pneumoniae. Pneumococcal growth was picked (3–5 colonies) from half of a plate, streaked on a sheep blood agar BA plate, and an oxacillin disc (1 µg; Biodisk, Sweden) was put on the densely streaked area. After overnight incubation in a candle jar an inhibition zone ≥ 20 mm around the oxacillin disc indicated penicillin sensitivity. Samples from the area of dense growth on the plate were collected with three alginate swabs. One was used for serotyping and two were used for storage in tubes containing STGG medium in two separate freezers (–20°C and –40°C).8,9

Serotyping of S. pneumoniae. Serotyping at GVRL was performed using the chessboard modification of the quellung method in which two sets of pneumococcal antiserum pools (A through I and P through S) and omniserum (Statens Seruminstitut, Copenhagen, Denmark), but no group- or type-specific antisera, were used.4 For serotyping, fresh overnight growth was collected with a calcium alginate swab and placed in a tube of brain heart infusion broth. The tube was incubated for 4–5 hours at 37°C and three drops of formalin were added. This pneumococcal suspension was used with antiserum pools in the quellung test. Twenty-one pneumococcal serogroups or serotypes were obtained from the combined reactions to pools A through I and P through T (Table 1Go). Isolates that reacted with only one of pools A through I but with none of pools P through T were identified only to the pool level. Isolates that did not react with any of the pools but reacted with omniserum were classified as non-typeable and those with no reactivity to antisera as non-capsulated (rough).


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TABLE 1
Chessboard system for typing and/or grouping of most pneumococci isolated from blood or cerebrospinal fluid*{dagger}
 
For serotyping at KTL, isolates stored usually for less than half a year (maximum = 13 months) at GVRL were transported on dry ice to Oulu, Finland and stored at –70°C until used. They were then streaked onto sheep blood agar plates. Serotyping was performed using counterimmunoelectrophoresis and latex agglutination (for group 7 and type 14) as the basic methods. The quellung method was used when needed for confirmation of equivocal results, as previously described.7 KTL-Oulu has participated in a study to evaluate the quality of serotyping of pneumococci in Europe.10 All antisera were obtained from the Statens Seruminstitut. For isolates that could not be used (17.3%), serotyping of isolates identified at GVRL as group 6, 19, or 23 was performed directly from a thawed tube of STGG medium, an approach that identifies the same serogroup/type as typing from cultured colonies (KTL-Oulu, unpublished data).

Data management and analysis. The bacteriologic database (EpiInfo version 6; Centers for Disease Control and Prevention, Atlanta, GA) of serotyping results obtained at GVRL was complemented with results obtained at KTL. Results obtained at GVRL and KTL were compared using SPSS version 11 (SPSS Inc., Chicago, IL) and Microsoft Excel® (Microsoft, Redmond, WA). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for the serotyping results obtained at GVRL, with results obtained at KTL used as the gold standard. The results obtained at KTL were considered the final results in further epidemiologic analyses. In cases in which the isolates were not viable (168) in or not sent (five) to KTL, the results obtained at GVRL were taken as final (Granat S, Zakaria MM and others, unpublished data).


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Streptococcus pneumoniae was isolated at GVRL from 1,274 of 4,324 nasopharyngeal samples from 99 index children, their mothers, and other family members. All isolates were serotyped using the chessboard quellung method at GVRL and 1,269 isolates sent to KTL-Oulu for confirmation of species identification and serotype/group. Of these, 1,048 (82.3%) were viable when received in Oulu. They were cultured, identified, and serotyped using the combined latex agglutination and counterimmunoelectrophoresis method in use at KTL.7 Two hundred twenty-one isolates were not viable when they reached KTL-Oulu, most probably because of an accidental thawing for several days in the freezer in GVRL. Of these, those 53 identified at GVRL as serogroup 6, 19, or 23 were analyzed by typing and subtyping directly from a tube of STGG medium. We have previous experience at KTL-Oulu in successful typing by this method and encountered no difficulties. Thus, we have results from both GVRL and KTL-Oulu for 1,101 isolates (86.4% of the 1,274 typed in GVRL). These 1,101 were used for comparison of the two laboratories and methods used.

Comparisons for the most common serogroups (6, 19, and 23) and the sensitivity, specificity, PPV, and NPV are shown in Table 2Go. The sensitivity of the chessboard method for correctly identifying serogroup 6, 19, and 23 isolates ranged from 86.5% (group 6) to 95.7% (group 23), and the specificity ranged from 97.9% (group 23) to 99.4% (group 19). The sensitivity was lower in identifying group 6 (86.5%) isolates in comparison with group 19 (91.5%) and group 23 (95.7%) isolates because 16 (61%) of the 26 group 6 isolates not identified at GVRL were classified as non-typeable. Since the chessboard method does not distinguish between serotypes within groups, the isolates of groups 6, 19, and 23 were subtyped only at KTL-Oulu. We compared the sensitivity of the chessboard method separately for serotypes 6A versus 6B, 23F versus 23A, and 19F versus 19A without detecting obvious differences according to the subtype (Table 2Go).


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TABLE 2
Sensitivity (SEN), specificity (SPE), positive predictive value (PPV), and negative predictive value (NPV) for identifying of serogroups 6, 19, and 23 among 1,101 nasopharyngeal Streptococcus pneumoniae isolates at GVRL by the chessboard method (Sørensen4) compared with conventional serotyping at KTL-Oulu*
 
Comparison of GVRL and KTL-Oulu results for the 10 most common serogroups/types and isolates reacting only with pool G antiserum are shown in Table 3Go. Pool G includes serotypes 29, 34, and 42 and groups 35 and 47, and can be identified by the chessboard method only to the pool level. For each of these groups and the pool G isolates, both the sensitivity (85.1–100%) and the specificity (97.9–99.9%) were very good. In addition, good sensitivity (77.8–94.4%) and specificity (98.7–99.9%) values were also observed for less common (10–20 isolates each) serogroups 9 and 17 and serotype 20. Of the uncommon (less than 10 isolates each) serotypes/groups, types 2, 5, and 8 were identified with a sensitivity and specificity of 100%, whereas for type 3 and group 12 these values were lower but still good. Of the three type 1 isolates, GVRL correctly identified only one.


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TABLE 3
Sensitivity (SEN), specificity (SPE), positive predictive value (PPV), and negative predictive value (NPV) for identifying of the 10 most common serogroups/types (6, 19, 15, 23, 33, 14, 11, 7, and 18) and those reacting with pool G antisera among 1,101 nasopharyngeal Streptococcus pneumoniae isolates serotyped at GVRL by the chessboard method (Sørensen4) compared with conventional serotyping at KTL-Oulu*
 
Among the 1,101 isolates identified and serotyped at KTL-Oulu, only 18 (1.6%) could not be confirmed as pneumococci. Of the 69 isolates identified at GVRL as non-typeable, 27 (39.1%) were confirmed as non-typeable, 10 (14.5%) were not pneumococci, and the remaining 32 (46.4%) were typeable pneumococci, among which 16 belonged to group 6 and four to type 34 in pool G; the rest were single isolates of different serogroups/types.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, the chessboard method of serotyping was introduced successfully into a small laboratory having very little experience with pneumococcal bacteriology in general and no experience in pneumococcal serotyping.4 The results were very satisfactory and would have well given the correct picture pneumococcal colonization as well as of distribution of serogroups/types in surveillance and epidemiologic studies. We believe that the important points in the success of this project were the initial bench-side training of the personnel, subsequent continuous surveillance and support of the study group, and the quality assurance of the reference laboratory, points that we have found important in an earlier project in The Philippines.11

The serotyping results compared with those obtained in a national reference laboratory showed very good sensitivity (86–100%) and specificity (98–100%) for the 10 most common serogroups/types (> 20 isolates). In addition, less common and even rare serogroups/types were usually identified with good sensitivity and specificity. However, an inherent limitation of the chessboard method is that it does not distinguish between serotypes within groups, which may be of importance when evaluating vaccine effects. Conversely, in most cases subtyping of only a few common groups may be needed, and this could be easily incorporated in the procedure by adding the required specific factor sera.

This approach has been applied in a collaborative acute respiratory infections vaccine (ARIVAC) project at the Research Institute for Tropical Medicine (RITM) in Manila, the Philippines, with KTL-Oulu as the technical support laboratory. At the RITM, the chessboard method using pools (A–I and P–T) was supplemented with a selection of group- or type-specific and factor antisera used to confirm the result and to identify subtypes of group 6, 7, 9, 18, 19 and 23 isolates. Ten percent of the isolates have been sent to the KTL for quality assurance, and the results at RITM and those at KTL-Oulu have been concordant in 90–95% of the isolates (Nissinen A and others, unpublished data). In addition, all problematic or ambiguous isolates, mostly non-typeable or those reacting only with pool G antiserum, were sent to KTL-Oulu for confirmation. In the present study, the isolates identified at GVRL as non-typeable were often typeable (46.4%) or not pneumococci (14.5%), which emphasizes the need for confirmation of such isolates in a reference laboratory.

In previous evaluations of methods using the chessboard principle with antiserum pools A–I and P–T in comparison to conventional typing, both methods have been used in the same established laboratories,5,6 whereas in the present study and also in the ARIVAC project in The Philippines, the field laboratory has had quite limited (RITM) or no (GVRL) earlier experience in serotyping and was located far from the support laboratory. The latter has meant the need of storing, transporting, and reviving of the isolates, all steps that can be sources of different types of errors. Thus, the present study, combined with the experience in the Philippines, is important as a real field evaluation of the chessboard method.

In the present study, a large number of non-selected nasopharyngeal isolates were studied. However, in the study of Lalitha and others5 only invasive isolates were serotyped, and in the study of Slotved and others6 only selected strains from various sources were serotyped. There is a considerable percentage of non-typable, non-capsulated pneumococci in the nasopharynx but few if any among the blood or cerebrospinal fluid (CSF) isolates.12–16 Experience in our laboratory (KTL-Oulu) suggests that the invasive blood or CSF isolates show more clear cut results in serotyping than the isolates from nasopharyngeal carriers. There are also {alpha}-hemolytic streptococci in the nasopharynx that resemble pneumococci but are difficult to identify.17 Thus, the origin of the isolates should also be considered when evaluating serotyping methods and laboratory performance. Originally, the chessboard method was intended for typing and/or grouping of most pneumococci isolated from blood or CSF.4

The reliability of the chessboard principle in serotyping of S. pneumoniae, which uses 12 pooled antisera (A–I, P–T), has been demonstrated in two recent studies. Lalitha and others compared serotyping by coagglutination with 12 pooled antisera (chessboard method) to serotyping by coagglutination with 55-antiserum panel with a good concordance (96.4%).5 In a recently developed kit for serotyping of pneumococci (Pneumotest-Latex, Statens Seruminstitut), the antiserum pools are applied to latex particles.6 In a blind test of 352 isolates of different origins, 336 (95.5%) were typed or grouped correctly by Pneumotest-Latex and positive latex agglutination reactions were easier and more rapid to interpret than the quellung reaction with a microscope.6

In conclusion, our study demonstrates that serotyping of pneumococci by the chessboard method can be introduced into a small laboratory with only basic bacteriologic skills provided adequate initial training, continuous external support, and qualified, responsible local supervision are available. In our study on the development of pneumococcal carriage in newborn children in Bangladesh, we used these serotyping results to confirm and subtype (groups 6, 19, and 23) at KTL-Oulu as described (Granat S, Zakaria MM and others, unpublished data). The results in this report also show that the original results obtained at GVRL would have been useful and reliable enough for detecting among the isolates the serogroups/types included in the 23 valent polysaccharide vaccine. Other groups/types could be identified only to the pool level, but these are considerably less common, except those reacting with the pool G where type 34 was quite common. The chessboard modification does not give the subtypes within the groups included (6, 7, 9, 10, 11, 12, 15, 17, 18, 19, 22, 23, and 33), but the subtyping can be done if needed with the same quellung method by adding testing with specific factor antisera. Pneumotest-Latex may be a promising alternative for the chessboard quellung method for serotype/group determination of pneumococci outside reference laboratories.6 However, there are as yet no latex reagents available for subtyping within the groups.

Received April 18, 2005. Accepted for publication August 29, 2005.

Acknowledgments: We thank Hasina Ferdous, Aesha Khatun, Ismat Ara, and Rokeya Parveen (GVRL), and Katriina Autio and Tarja Kaijalainen (KTL-Oulu) for excellent laboratory work, and Dr. Milan Das and Dr. Mokidur Rahman for organizing the collection of nasopharyngeal secretion samples.

Financial support: This study was supported by the European Commission DG Research INCO Programme (Contract ICA4-1999-10008).

Disclosure: None of the authors has any conflicts of interests.

* Address correspondence to Elja Herva, National Public Health Institute, Box 310 FIN-90101, Oulu, Finland. E-mail: elja.herva{at}ktl.fi Back

Authors’ addresses: Elja Herva, National Public Health Institute, Box 310 FIN-90101, Oulu, Finland, Telephone: 358-8-537-6210, Fax: 358-8-537-6251, E-mail: elja.herva{at}ktl.fi. Simo Granat, Jukka Ollgren, Liisa Piirainen, and P. Helena Mäkelä, National Public Health Institute, Mannerheimintie 166, FIN-00300, Helsinki, Finland, Telephone: 358-9-47441, Fax: 358-9-4744-8675, E-mails: simo.granat{at}ktl.fi, jukka.ollgren{at}ktl.fi, liisa.piirainen{at}ktl.fi, and pirjo.makela{at}ktl.fi. Zakaria Mia, Gonoshasthaya Vaccine Research Laboratory, Savar, Dhaka 1344, Bangladesh, Telephone: 880-2-770-8856, Fax: 880-2-770-8316, E-mail: mmzakaria{at}yahoo.com.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bogaert D, de Groot R, Hermans PWH, 2004. Streptococcus pneumoniae colonisation: the key to pneumococcal disease. Lancet Infect Dis 4: 144–154.[ISI][Medline]
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  7. Kilpi T, Herva E, Kaijalainen T, Syrjänen R, Takala AK, 2001. Bacteriology of otitis media in a cohort of Finnish children followed for the first two years of life. Pediatr Infect Dis J 20: 654–662.[ISI][Medline]
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