MIMET-04598; No of Pages 9 Journal of Microbiological Methods xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth

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Review

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Current methods for capsular typing of Streptococcus pneumoniae

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Article history: Received 21 November 2014 Received in revised form 21 February 2015 Accepted 8 March 2015 Available online xxxx

16 17 18 19

Keywords: Streptococcus pneumoniae Capsular polysaccharide Serotyping

Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton SO16 6YD, UK Infectious Diseases, Genome Institute of Singapore, Singapore 138672, Singapore NIHR Southampton Respiratory Biomedical Research Unit, University Hospital Southampton NHS Foundation Trust, Southampton SO16 6YD, UK d NIHR Wellcome Trust Clinical Research Facility, University Hospital Southampton NHS Foundation Trust, Southampton SO16 6YD, UK e London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK b

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Elita Jauneikaite a,b,1, Anna S. Tocheva a,1, Johanna M.C. Jefferies a,c, Rebecca A. Gladstone a, Saul N. Faust a,c,d, Myron Christodoulides a, Martin L. Hibberd b,e, Stuart C. Clarke a,c,⁎

Streptococcus pneumoniae is a major respiratory tract pathogen causing pneumococcal disease mainly in children aged less than five years and in the elderly. Ninety-eight different capsular types (serotypes) of pneumococci have been reported, but pneumococcal conjugate vaccines (PCV) include polysaccharide antigens against only 7, 10 or 13 serotypes. It is therefore important to track the emergence of serotypes due to the clonal expansion of non-vaccine serotypes. Increased numbers of carried and disease-causing pneumococci are now being analysed as part of the post-PCV implementation surveillance studies and hence rapid, accurate and costeffective typing methods are important. Here we describe serotyping methods published prior to 10th November 2014 for pneumococcal capsule typing. Sixteen methods were identified; six were based on serological tests using immunological properties of the capsular epitopes, eight were semi-automated molecular tests, and one describes the identification of capsular type directly from whole genome data, which also allows for further intra and inter-genome analyses. There was no single method that could be recommended for all pneumococcal capsular typing applications. Although the Quellung reaction is still considered to be the gold-standard, laboratories should take into account the number of pneumococcal isolates and the type of samples to be used for testing, the time frame for the results and the resources available in order to select the most appropriate method. Most likely, a combination of phenotypic and genotypic methods would be optimal to monitor and evaluate the impact of pneumococcal conjugate vaccines and to provide information for future vaccine formulations. © 2015 Published by Elsevier B.V.

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Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . Phenotypic methods . . . . . . . . . . . . 2.1. Quellung reaction . . . . . . . . . . 2.2. Co-agglutination and latex agglutination 2.3. Immunoblot assays. . . . . . . . . . 2.4. ELISA and EIA . . . . . . . . . . . . 2.5. Flow cytometry . . . . . . . . . . . 2.6. Genotypic methods . . . . . . . . . 2.7. Multiplex PCR (mPCR) . . . . . . . . 2.8. PCR-RFLP . . . . . . . . . . . . . . 2.9. FAF-mPCR . . . . . . . . . . . . . . 2.10. PCR/ESI-MS . . . . . . . . . . . . 2.11. mPCR/RLB . . . . . . . . . . . . .

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⁎ Corresponding author at: Infectious Disease Epidemiology Group, Mailpoint 814, Level C, Sir Henry Wellcome Laboratories, South Block, University Hospital Southampton NHS Foundation Trust, Southampton SO16 6YD, UK. E-mail address: [email protected] (S.C. Clarke). 1 These authors contributed equally to the work.

http://dx.doi.org/10.1016/j.mimet.2015.03.006 0167-7012/© 2015 Published by Elsevier B.V.

Please cite this article as: Jauneikaite, E., et al., Current methods for capsular typing of Streptococcus pneumoniae, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.03.006

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E. Jauneikaite et al. / Journal of Microbiological Methods xxx (2015) xxx–xxx

2.12. Real time PCR (RT-PCR) . . . . . . . . . . . . . . 2.13. Nanofluidic Real Time PCR . . . . . . . . . . . . . 2.14. Microarray . . . . . . . . . . . . . . . . . . . . 2.15. Sequetyping . . . . . . . . . . . . . . . . . . . 2.16. Target enrichment based next generation sequencing . 2.17. Serotype inference from whole genome sequencing . 3. Discussion . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

2. Phenotypic methods

69 Q4 70

Streptococcus pneumoniae (the pneumococcus) causes ~ 500,000 deaths of children under the age of five years annually, primarily in developing countries (WHO, 2012). There are ninety-eight reported pneumococcal serotypes classified on biochemical and genetic differences in the structure of the capsular polysaccharide (CPS), including the novel serotypes 6D (Jin et al., 2009), 6F and 6G (Oliver et al., 2013), 11E (Calix and Nahm, 2010), 20A and 20B (Calix et al., 2012) and putative serotype 6E (Ko et al., 2013), which are grouped into 46 serogroups based on their antigenic similarities (Henrichsen, 1995). The specific distribution of pneumococcal serotypes and serogroups has been associated with age, site of infection, pre-existing medical conditions, social status and geographic region (Hausdorff et al., 2000a,b, 2005). Reports suggest that the biochemical properties of the CPS are predictors for serotype specific carriage prevalence, growth and invasive pneumococcal disease (IPD) potential (Brueggemann et al., 2003; Hathaway et al., 2012; Sjostrom et al., 2006; Weinberger et al., 2009). The majority of IPD in adults and children as well as acute otitis media in children under five years of age can be attributed to only 11 serogroups (Hausdorff et al., 2000b; Johnson et al., 2010). The accurate identification of pneumococcal serotypes is paramount for disease surveillance and pre- and post-pneumococcal vaccine evaluation. In the UK and other countries, the emergence of non-vaccine serotypes after PCV7 and PCV13 implementations has been reported in carriage and disease. Increases in carriage of serotypes 6C, 33F and 22F were reported in the paediatric population (Tocheva, 2011; Tocheva et al., 2013; van Hoek et al., 2014). Changes in disease-causing serotypes after pneumococcal vaccine implementations included the decrease of 19A, but increase in serotypes 15A and 15B, 35B, 23B and 6C (Jefferies et al., 2010; Hanage et al., 2011; Miller et al., 2011; Rosen et al., 2011; Richter et al., 2014; Regev-Yochay et al., 2015). While pneumococcal capsular typing uses antisera to detect serogroup and serotype-specific pneumococcal capsule epitopes, molecular techniques identify the pneumococcal serotypes based on the nucleotide sequence of the capsule gene. Here, we review studies published on pneumococcal capsular typing methods identified using the PubMed database before 10th November 2014. Search terms used to identify relevant articles were: [S. pneumoniae], [pneumococcus], [capsule type identification], [serotype identification] and [serotyping methods]. Further studies were identified from the reference lists of primary studies. Only studies published in English and with available full text articles were used in this review. To provide a convenient summary of strengths and limitations of the various methods available, we used the following criteria: sensitivity, specificity, number of serotypes that can be identified by the method, approximate costs, time required to prepare the samples and perform a test, level of training required to carry out the method, shelf life of reagents, possibility of using the method for batch processing, detection of multiple serotypes within the same sample and direct pneumococcal serotype detection from clinical samples (Table 1).

2.1. Quellung reaction

88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117

118 119 120 121

2.2. Co-agglutination and latex agglutination

141

The development of agglutination tests reduced the time required to serotype a batch of pneumococcal isolates (Kirkman et al., 1970). A fresh culture of pneumococci is mixed with type specific serum raised against 1 of 46 pneumococcal serogroups in each well of a microtitre plate. The agglutination reaction that occurs between the pneumococcus and the type-specific antibodies in the serum results in visible clumping eliminating the requirement for microscopy (Kirkman et al., 1970). This method was improved by incorporating factor-specific antiserum and carried out on a microscope slide, i.e., slide agglutination test (Kronvall, 1973). Commercially available omni antiserum enables the detection of 91 of the 98 reported pneumococcal serotypes with this method (Christensen et al., 1973; Kronvall, 1973; Lund and Rasmussen, 1966; SSI, 2012). In 1986, the co-agglutination method was improved for pneumococcal typing from culture and clinical samples by binding formalin fixed staphylococcus protein A to a serotype specific (factor) antibodies rather than serogroup specific antibodies as done previously, thus minimizing cross-reactivity (Smart, 1986; Smart and Henrichsen, 1986). The chess-board system for typing pneumococci was introduced in 1993 and served as the basis for the Pneumotest-latex test developed by the Statens' Serum Institute. It identifies the 23 serotypes included in the 23-valent pneumococcal polysaccharide vaccine and additional 25 cross-reacting types (Table 2). This method was later improved by introducing the latex agglutination test for identification and capsular typing of pneumococci, which allowed pneumococcal identification

142 143

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The Quellung reaction (Neufeld, 1902; Sorensen, 1993) is still considered the gold-standard for pneumococcal serotyping (Austrian, 1976). It is reported to be highly sensitive and specific and remains the method against which all other methods are compared (Satzke et al., 2013). It is an in-situ immunoprecipitation method whereby rabbit antiserum is added to the pneumococcal suspension. If the antibodies recognise a specific capsule epitope, they bind to the cell wall and produce a change in the refractile index of light passing through the capsule, which appears swollen under a microscope (Sorensen, 1993). One limitation of the Quellung reaction is that the test is unable to detect multiple serotypes within the same sample (Kaijalainen, 2006). It is also inconvenient for serotyping large number of isolates and is relatively laborious and costly as it requires a supply of pneumococcal antisera. To improve the latter, Habib and colleagues (Habib et al., 2014) developed an improved Quellung protocol that minimizes the amount of antisera used (Habib et al., 2014) (a video for the protocol is also available at http://www.jove.com/video/51208/). This is done by testing 1 μl of pneumococcal suspension against pools made up from the most common antisera and testing the sample against only those individual antisera from the pools that give a positive result (Habib et al., 2014).

T

86 87

C

84 85

E

82 83

R

81

R

79 80

O

77 78

C

75 76

N

73 74

U

71 72

O

68

F

67

Please cite this article as: Jauneikaite, E., et al., Current methods for capsular typing of Streptococcus pneumoniae, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.03.006

122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140

144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166

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N

Table 1 A comparison of pneumococcal capsular typing methods.

t1:3

C

Phenotypic

t1:4

Criteria

t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11

n/a Specificity (compared to conventional methods) No. of 91 serotypes

t1:12

Cost

Genotypic

Quellung Co/Latex Colony/dot ELISA Flow reaction agglutination blot assay cytometry

B+D

O

Luminex/ Bio-plex

R

n/a

96%

98%

100%

n/a

91

15

23

23

23

B

B

B

B+D

B+D

Multiplex PCR

PCR-RFLP FAF-mPCR

PCR/ESI-MS mPCR/RLB RT-PCR Nanofluidic Micro Sequetyping Targeted-enriched Whole RT-PCR array NGS genome inference

100%

n/a

99.2%

84.7%

71%

98.6%

95.7%

95.2%

86%

97%

96%

40

45

23

35

50

43

48

38

A+D

A+D

A+D

A+D

A+D

A+

A+D

A+D

94 (plus identify new serotypes) C+D

1–2 days

2–4 days

R 46

E

A+D

46

C

A+D

1 day

2–3 h

1–2 days

D 1 day

1–2

1–2

2

1–2

1–2

1–2

1–3

3–14 days (depending on the number of samples) 1–5

1 yr +

1 yr +

1 yr +

1 yr +

1 yr +

1 yr +

6 mth–1 yr

6 mth–1 yr

Yes

Yes

Yes

Yes

Yes

1 yr + Yes

Yes

Yes

Yes

Potentially Potentially Potentially No yes yes yes

Potentially Yes yes

Yes

Yes

Potentially Potentially yes yes

Yes

T

b1 day

t1:13 t1:14 t1:15 t1:16

Time required for sample preparation and testing

1–2 days

1 day

1 day

1 day 1 day

1 day

2–3 h

1 day

t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29

Level of training Shelf life of reagents Batch processing Detection of multiple serotypes Direct detection from clinical sample

2–3

2–3

2–3

2

1–2

1–2

1

1–2

1–2

3 yrs

3 yrs

1 yr +

1 yr +

1 yr +

1 yr +

1 yr +

No

No

No

1 yr + Yes

Yes

Yes

Yes

Yes

No

No

Yes

No

No

No

Yes

Yes

t1:30 t1:31 t1:32 t1:33 t1:34

1 day

E

Yes

Potentially No yes

D

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R O Yes

Yes

Yes

n/a

Potentially yes

Potentially yes

Yes

Potentially yes

Yes

No

Yes

No

E. Jauneikaite et al. / Journal of Microbiological Methods xxx (2015) xxx–xxx

Please cite this article as: Jauneikaite, E., et al., Current methods for capsular typing of Streptococcus pneumoniae, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.03.006

t1:1 t1:2

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F

Information summarized in this table was taken from published articles that are referenced in this review and from personal experience of the authors. Reported sensitivity and specificity values are included in the table. n/a — no information was available. Costs vary between different countries and companies, therefore, here we classed costs as A b B b C. Costs: A — molecular reagents such as primers and polymerases; B — biological reagents such as polysaccharides, antibodies; C — next Generation Sequencing requires DNA library building kits and other equipment; D — specialized equipment: microscope, PCR machine or sequencer etc. Level of training: 1 — basic experience in microbiology and molecular biology; 2 — well trained/ experienced staff; 3 — Specialized knowledge in programming or/and background in bioinformatics. Time required for sample preparation and testing is approximate and can vary depending on the number of samples and the experience of the person and does not include time required to prepare special reagents (for e.g., conjugating antibodies to carboxyl beads) or isolate/identify pneumococcus.

3

4 t2:1 t2:2 t2:3 t2:4 t2:5

E. Jauneikaite et al. / Journal of Microbiological Methods xxx (2015) xxx–xxx

Table 2 The chessboard system for pneumococcal capsular type identification. The chessboard system was developed for the fast and accurate identification of the most common pneumococcal serogroups, causing the majority of pneumococcal disease. Pools A-I were first developed by the Statens Serum Institute, and pools P-T were later included in the system to identify the serotypes included in the 23-valent polysaccharide vaccine. Pools P-T also identifies all the serotypes currently included in the pneumococcal conjugate vaccines. This typing system identifies all 21-vaccine groups and the additional 25 serogroups, not included in any currently available pneumococcal vaccines.

t2:6

Pool

P

Q

R

S

T

t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15

A B C D E F G H I

1* 19(F*, A*,B, C) 7(F*, A, B, C)

18(A,B,C*,F) 6(A*, B*, C)

4* 3*

5* 8

2

9(A, L, N, V*) 12(F, A, B)

23(F*, A, B)

20 11(F, A, B, C, D) 33(F, A, B, C, D) 22(F, A)

24(F, A, B), 31, 40 16(F, A), 36, 37 21, 39 27, 32(F, A), 41(F, A) 29, 34, 35(F, A, B, C), 42, 47(F, A) 13, 28(F, A) 25(F, A), 38, 43, 44, 45, 46, 48

15(F, A, B, C)

F

14*

10(F, A, B, C) 17(F, A)

Non-vaccine types/groups

Serotypes/serogroups in bold are included in the 23-valent pneumococcal polysaccharide vaccine. (*) indicates serotypes included in the conjugate vaccine formulations: PCV7 (serotypes 4, 6B, 9V, 14, 18C, 19F and 23F); PCV10 (PCV7 + serotypes 1, 5 and 7F) and PCV13 (PCV10 + serotypes 3, 6A and 19A). Adapted from Sorensen et al. (Sorensen, 1993) and Slotved et al. (Slotved et al., 2004).

167

170

directly from cerebrospinal fluid (Coonrod and Rylko, 1976; Slotved et al., 2004). The described agglutination method is a break-through due to its accuracy, rapidity and the significant reduction of antiserum used compared to the Quellung reaction (Porter et al., 2014).

171

2.3. Immunoblot assays

172 173

187

Dot blot (Fenoll et al., 1997) and colony blot assays (Bogaert et al., 2004) have been suggested as alternatives to the Quellung reaction as faster and less expensive methods for pneumococcal typing. In both assays, pneumococci are blotted on nitrocellulose membranes, which are incubated with capsule-specific rabbit antisera and positive reactions are visualised with horseradish peroxidise-conjugated anti-rabbit immunoglobulin G (IgG) in the presence of a chemical substrate. The main difference between the two assays is that the colony blot assay identifies a minor serotype in a primary pneumococcal culture among predominant serotypes (Bogaert et al., 2004). However, a high proportion of false positive results in the dot blot assay using polyclonal rabbit antisera led to the implementation of mouse serotype-specific monoclonal antibodies (Bronsdon et al., 2004). The immunoblot assays allow for batch processing of pneumococci and detection of minor serotypes present in the same sample, but remain time-consuming for field epidemiological pneumococcal typing.

188

2.4. ELISA and EIA

189 190

202

An enzyme-linked immunosorbent assay (ELISA) and a competitive enzyme immunoassay (EIA) have also been developed for detection of pneumococcal CPS from serum, nasopharyngeal swabs and urine (Lankinen et al., 2004; Leeming et al., 2005; Schaffner et al., 1991). These assays require high antibody titres to improve avidity and have been demonstrated to detect pneumococcal antigen with high sensitivity. To overcome the issue of low antibody titres in commercially available antisera, the CPS-specific antiserum is raised in-house in rabbits. However, while those methods showed high sensitivity of CPSdetection from serum and urine, and are simpler compared to the conventional culture and Quellung reaction, the CPS-specific antisera are not commercially available, and the ELISA and EIA assays are time consuming, the results are available after several days and require the use of reagents with limited shelf life.

203

2.5. Flow cytometry

204 205

Flow cytometry enables the separation of cells and microparticles from a complex mixture based on their size, granularity and fluorescent labels (Kellar and Iannone, 2002). The microspheres used in published flow cytometry methods for pneumococcal serotyping have different sizes and intensities of red fluorescence, which allow different

185 186

191 192 193 194 195 196 197 198 199 200 201

206 207 208

209

2.6. Genotypic methods

238

The nucleotide sequences of the capsular loci of 90 pneumococcal serotypes were reviewed in 2006 (Bentley et al., 2006). Information on serotype-specific genes within these CPS loci has allowed the development of genotypic assays to determine the presence of pneumococcal capsule genes encoding specific capsular types using different molecular techniques.

239 240

2.7. Multiplex PCR (mPCR)

245

A PCR method was developed for the simultaneous detection of capsule specific genes of 40 pneumococcal serotypes (Brito et al., 2003; Pai et al., 2006). The current multiplex PCR (mPCR) assay allows sequential use of PCR primer pools with the most common disease-causing serotypes (Dias et al., 2007; Morais et al., 2007; CDC). This allows the negative elimination of serotypes in order to reduce time and reagent used (Pai et al., 2006). There are eight pools of primer pairs (plus an

246 247

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microspheres bound to various antigens or antibodies to be pooled together and tested on one sample simultaneously (Kellar and Iannone, 2002; Park et al., 2000; Yu et al., 2005). Microsphere technology assays were adapted for use in the Luminex® multiple analytes profiling system (Luminex, USA) and BioPlex® multi-analyte suspension array instrument (Bio-Rad, USA) to detect serotype-specific pneumococcal polysaccharides in clinical samples (competitive inhibition principle) (Findlow et al., 2009; Sheppard et al., 2011). The method uses colour-coded Luminex xMAP® microparticles that are coated with monoclonal cps-specific antibodies. Following an overnight incubation with the test samples, a polyclonal rabbit serum is added and the reaction is developed with phycoerythrin-conjugated anti-rabbit antibody. Another assay, a competitive inhibition flow analysis, is based on the conjugation of pneumococcal polysaccharides rather than monoclonal antibodies to the beads (Findlow et al., 2009; Lal et al., 2005; Pickering et al., 2002). Free antigens in the sample compete with antigens conjugated to the beads for binding to the antibody. Currently, this method enables the detection of PCV serotypes from bacterial lysates (Park et al., 2000; Yu et al., 2005) and directly from clinical samples (Findlow et al., 2009; Sheppard et al., 2011). The flow cytometry and microsphere technologies allow rapid and accurate capsular typing for a batch of pneumococcal isolates and detection of serotypes from small volumes of clinical sample. There is a theoretical possibility of detecting multiple serotypes, but this would require further studies to improve the assays. Though flow cytometry based assays use fewer reagents per sample and small volumes of the clinical samples. Instrument purchase and regular instrument maintenance both add to the overall costs of using these methods.

T

C

E

183 184

R

181 182

R

179 180

O

177 178

C

175 176

N

174

U

168 169

O

t2:16 t2:17 t2:18

Please cite this article as: Jauneikaite, E., et al., Current methods for capsular typing of Streptococcus pneumoniae, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.03.006

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2.9. FAF-mPCR

288

300 301

A high-throughput method was developed combining the mPCR fragment analysis and automated fluorescent capillary electrophoresis (FAF-mPCR) using a genetic fragment size analyser (3130xl, ABI) (Selva et al., 2012) to detect serotypes. PCR primers are fluorescently labelled to provide a high sensitivity for amplified fragment detection (Selva et al., 2012). Following mPCR, amplified products are separated using capillary electrophoresis and the data are analysed based on the fluorescent peaks according to the specific colour and size assigned to the corresponding pneumococcal serotype (Selva et al., 2012). The main limitation of this method is the requirement for the automated fluorescent fragment size analyser that might be expensive. Advantages of this method include a lower cost than traditional phenotypic serotyping methods, rapidity and the capacity to detect secondary serotypes in mixed cultures.

302

2.10. PCR/ESI-MS

303

A method combining serotyping PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) has been developed. This uses an electrospray ionization mass spectrometer (Ibis T5000) to analyse base composition of the serotype amplicons (Massire et al., 2012). Sample DNA is distributed into each well of a microtitre plate together with a set of serotype specific and a set of internal positive control primers (Massire et al., 2012). Following amplification, PCR products are electrosprayed into a mass spectrometer and the base composition of each DNA fragment determined based on the mass/charge ratio (Massire et al., 2012). Molecular signatures of amplified products from

279 280 281 282 283 284

289 290 291 292 293 294 295 296 297 298 299

304 305 306 307 308 309 310 311 312

C

278

E

276 277

R

274 275

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Another typing method combining mPCR and reverse line blot hybridization assay (mPCR/RLB) allows the identification of pneumococcal capsular types omitting the gel electrophoresis step (Kong et al., 2006) by utilising serotype-specific DNA probes (O'Sullivan et al., 2011). Biotin labelled PCR products are hybridized to the DNA probes on a nylon membrane, and detected using peroxidase-labelled streptavidin. Peroxidase acts as a catalyst in a light-producing reaction to which a light sensitive film is exposed (O'Sullivan et al., 2011). The pattern on the film is examined and serotypes assigned according to the pattern when compared to a positive control. Originally the method was designed to identify the 23 serotypes included in the 23-valent pneumococcal polysaccharide vaccine — PPV23 (Kong et al., 2006). Primers and probes were later described for an additional 50 serotypes, allowing the identification of 90 pneumococcal serotypes (Zhou et al., 2007). The advantages of this method are the potential to recognise multiple serotypes present in the same sample (Zhou et al., 2007), reduced processing time and the re-use of the nylon membrane, which reduces consumables costs required for the assay.

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To optimise pneumococcal capsular typing directly from culture negative clinical specimens, specific probes were designed for a real time PCR (RT-PCR) assay that detects ply, wgz and 16 capsular polysaccharide clusters and allows the identification of 35 serotypes (Tarrago et al., 2008). The assay was reported to be 90% sensitive for serotype detection in blood cultures (Resti et al., 2010) and culture-negative pleural fluid samples from children even after they had antibiotic treatment (Tarrago et al., 2008). Currently a sequential triplex RT-PCR method is available for PCV13 serotypes plus eight additional serotypes (Pimenta et al., 2013). RT-PCR is less time consuming than gel-based methods. The region specific RT-PCR protocols are available on CDC website (CDC). The process allows the batch analysis of pneumococcal isolates directly from clinical samples. However, it requires a RT-PCR machine and expertise in optimising such assays.

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A high-throughput quantitative and qualitative method using nanofluidic RT-PCR (Fluidigm Biomark HD System) has been described (Dhoubhadel et al., 2014b). A mixture of previously reported primers and newly designed primers were used to identify 50 serotypes of 29 serogroups (this included all conjugate and polysaccharide pneumococcal vaccine serotypes) (Dhoubhadel et al., 2014b). The main advantage of this method is that it provides qualitative and quantitative data for dominant and minor populations of pneumococcal serotypes present within the sample (Dhoubhadel et al., 2014a; Dhoubhadel et al., 2014b). However, primers for the molecular determination of the serotypes are not specific enough especially for certain closely related serotypes, for example, serogroup 6 members. Also, the costs are higher for the nanofluidic RT-PCR equipment when compared to common RT-PCR machines.

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A PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) assay, based on visualising digested PCR amplicon fragments, has also been developed (Batt et al., 2005). Firstly, the gene cassette encoding pneumococcal capsule is amplified using primers for dexB and aliA genes. The PCR amplicon then is digested with HinfI enzyme and the resulting fragments are analysed using gel electrophoresis (Batt et al., 2005). These RFLP patterns are then assigned to each of 46 known serotypes using Bionumerics software (Applied Maths, Austin, USA) (Batt et al., 2005). A serotype is assigned to the “unknown” sample if ≥90% similarity in the band pattern is observed (Batt et al., 2005). PCR-RFLP technique is simple and fairly cheap. However, this method uses nonserotype specific genes to distinguish between the serotypes and employs a limited number of serotype patterns, which may result in errors. Also as the results are image-based there may be difficulties comparing data between laboratories.

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test samples are compared to a database of signatures derived from known capsular loci (Massire et al., 2012). The technology was set up and tested to identify 45 different serotypes/serogroups of S. pneumoniae and to simultaneously identify the genotype (Massire et al., 2012). This method is potentially suited to detect mixed serotypes and serotypes from culture-negative clinical specimens, however, the electrospray mass spectrometer is required. Other advantages compared to the phenotypic serotyping methods are reduced time when compared to phenotypic typing and reagents required.

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additional pool for identifying serotypes 6C and 6D) each of which can distinguish between five serotypes through detection of differently sized amplicons by gel electrophoresis. A new primer pool for mPCR detecting PCV13 serotypes was also suggested (Coskun-Ari et al., 2012). The primer specificity in mPCR is suitable for pneumococcal serotype detection directly from clinical samples (Saha et al., 2008) and is currently a recommended pneumococcal typing assay by the Centers for Disease Control and Prevention (CDC). mPCR is a rapid and low cost method for processing batches of clinical pneumococcal isolates for characterising common regions specific to serotypes. Unfortunately, the development of additional primer pairs has been hampered by high sequence homology in the pneumococcal capsule region of some serotypes leading to incomplete characterisation of the isolates. Additionally, a pneumococcal capsular typing algorithm combining mPCR and serological reactions was described to overcome problems of incomplete serotyping for studies where less common serotypes are most likely to be found and which may be useful for studies of pneumococcal carriage (Miernyk et al., 2011).

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Leung et al. (2012) used in silico analysis of the sequence of pneumococcal capsule locus from 138 isolates of 48 different serotypes to design a single primer pair which was then used to develop a serotype differentiation PCR. The amplicons are sequenced and their sequences compared using BLAST (Leung et al., 2012), which resulted in 85% of assay sensitivity. Although, the method is simple, the targeted cpsB gene is not specific enough for typing larger datasets as the reported results were ambiguous (only 85% specificity, which is low when compared to other available methods).

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A target enrichment based next generation sequencing method was suggested as an alternative to whole genome sequencing (Liyanapathirana et al., 2014). Two PCR primer pairs were used; the first differentiates pneumococci from other streptococcal species (using lytA gene primers) and the second to target a specific capsular region up to 500 bp in length (Liyanapathirana et al., 2014). PCR products were enriched prior to sequencing on bench-top sequencer (Liyanapathirana et al., 2014). Using this method, 33 serotypes were identified (Ip et al., 2014; Liyanapathirana et al., 2014). Advantages include the potential to identify multiple serotypes, the ability to employ batch processing and to identify serotypes directly from clinical samples (Ip et al., 2014). However, access to a next generation sequencer is required along with bioinformatics expertise and the primers described lack the specificity to identify closely related serotypes.

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Sequencing the entire pneumococcal genome is becoming more affordable as next generation technologies increase in capacity and become more widely used (Croucher et al., 2009; Li et al., 2012; Marttinen et al., 2012). This provides an opportunity to analyse the entire capsular locus from whole genome sequence data using a number of bioinformatics approaches. Assemblies can be interrogated using published serotype-specific primer sequences resulting in the recovery of an in silico “product” sequence without the biological restraints (CDC; Gladstone et al., 2012). Furthermore the sequence itself can be examined in detail, confirming serotype-specific gene sequences and single nucleotide polymorphisms (SNPs) distinguishing the members of serogroups. As well as enabling the inference of serotypes that are ambiguously typed by utilising

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While the information on pneumococcal serotype might not directly benefit the clinical diagnosis or the immediate treatment of the patient, the knowledge of pneumococcal serotypes circulating in carriage and disease is required to correctly estimate the impact of pneumococcal vaccines locally and globally, and is needed by vaccine policy makers. The pneumococcus is known for its genetic diversity in the capsular region (Bentley et al., 2006) and ability to switch capsule (Croucher et al., 2011). Since 2006, when 90 pneumococcal capsular loci were sequenced and described (Bentley et al., 2006), eight new serotypes have been reported (of which at least one is still to be biochemically confirmed). These findings underline the diverse nature of the pneumococcus and inform us that much is still to be discovered. For example, the difference in some of the serotypes is based only on two mutations in the capsular region. Good examples are serotypes 18B and 18C, which are highly similar to 18F (Bentley et al., 2006; Mavroidi et al., 2007), but can be easily distinguished by their acetylation patterns (Mavroidi et al., 2007); and serotypes 19F and 19A, which have 22% divergence in wzy gene (Bentley et al., 2006) that leads to different polymerization links (Morona et al., 1999). The high similarity between the genes of similar serotypes suggests that non-serotype specific capsule genes are not appropriate targets for pneumococcal serotyping methods and that care is needed even when employing serotype-specific genes when these may vary by only a few bases. Good quality data on pneumococcal serotype epidemiology is important to provide evidence-based recommendations for the composition of increased valency or altered combinations of PCVs. To achieve these aims, serotyping methods need to provide accurate results with high assay sensitivity and specificity. Here, we have described and compared sixteen pneumococcal capsular typing methods including the use of whole genome sequence data (Table 1). The information provided should help the laboratory or research group in deciding which methods are suitable for their use, depending on the sample throughput, experience of the personnel and the available equipment. The Quellung reaction is still recognised as the gold standard technique for pneumococcal typing. The Pneumotest latex method developed by the Statens' Serum Institute has become the most widely used phenotypic pneumococcal capsular typing method. Ortika et al. (2013) suggested that if the agglutination reagents were prepared inhouse, this could reduce reagent costs leading to wider use (Ortika et al., 2013). However, stringent quality control needs to be in place to ensure that in-house reagents are comparable to the commercially available kits. Of all phenotypic typing methods, the Pneumotest latex can identify the most pneumococcal serotypes The search for improved and faster methods to serotype batches of pneumococcal isolates has resulted in the development of serotyping assays based on flow cytometry and multiplex bead-based (for example Luminex®) technologies. Multiplex bead-based assays can differentiate closely related serotypes. This type of methodology also offers the advantage of simultaneous pneumococcal identification and can detect the capsular type directly

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Microarray assay uses specific probes to construct a serotyping microarray that detects the highly variable glycosyltransferase genes wzx and wzy which contain serotype or serogroup specific regions (Tomita et al., 2011; Wang et al., 2007) and 12 polymorphisms to help assign 22 serotypes and 24 serogroups (Raymond et al., 2013). Labelled pneumococcal DNA is added to the array chip to bind the complementary sequence prior to the microarray run. After the microarray run, the signals and background intensities were analysed using a microarray scanner (Tomita et al., 2011; Wang et al., 2007). The use of GT probes demonstrates the potential of the microarrays to inform directly on the biochemical structure of the CPS in addition to differentiating serotypes. The microarray technology offers a rapid and accurate pneumococcal capsular identification. Additionally, this method has enabled the detection of multiple serotypes from a number of different sample types, including aural discharge from cases of otitis media and nasopharyngeal carriage samples (Turner et al., 2011). Relative abundances of different serotypes can also be detected (Newton et al., 2011; Turner et al., 2011). However, this method requires a microarray scanner and wellexperienced personnel.

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gene sequences for further discrimination, the use of additional ‘primers’ that target multiple serotype-defining genes can also easily allow the inference of specific serotypes based on the combination of allele types at multiple serotype defining loci for example within serogroups 6 and 11 (Bratcher et al., 2010; Calix et al., 2011; Park et al., 2007). Mapping of raw sequencing reads against reference capsular locus sequences has also been used as a method to infer serotype in pneumococcal studies (Croucher et al., 2011; Everett et al., 2012). Additionally, an in silico method was successfully applied to differentiate the non-typable pneumococci (Chewapreecha et al., 2014) using previously described dexB-aliA primers (Salter et al., 2012). Although, whole genome sequencing is becoming more affordable, it still requires experience in sample preparation for sequencing and importantly the bioinformatics expertise to extract and analyse data from a capsular loci.

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SNF receives support from the National Institute for Health Research funding via the Southampton NIHR Wellcome Trust Clinical Research Facility and the Southampton NIHR Respiratory Biomedical Research Unit. SNF, SCC and JMJ act as principal investigator for clinical trials and other studies conducted on behalf of University Hospital Southampton NHS Foundation Trust/University of Southampton that are sponsored by vaccine manufacturers but receives no personal payments from them. SNF, JMJ, MC and SCC have participated in advisory boards for vaccine manufacturers but receive no personal payments for this work. SNF, SCC and JMJ have received financial assistance from vaccine manufacturers to attend conferences. All grants and honoraria are paid into accounts within the respective NHS Trusts or Universities, or to independent charities. All other authors have no conflicts of interest.

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Pneumococcal infections remain a significant cause of morbidity and mortality globally with young children and the elderly at the highest burden of contacting pneumococcal infection. Since current pneumococcal vaccine formulation consists of pneumococcal polysaccharides from different pneumococcal serotypes conjugated to a carrier protein, pneumococcal capsular typing is important for pneumococcal vaccine development, vaccine efficacy trials, and post-vaccine surveillance. A variety of different phenotypic and genotypic serotyping methods are currently available. Although there may not be a single universal

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capsular typing method that is used by all laboratories, there are an increasing number of robust methods available. The aim of this review was not to make recommendations, but to provide a concise comparison of the currently available pneumococcal serotyping methods. Therefore, the decision on the most appropriate method to use will be based on the nature of the scientific question, the number of bacterial isolates or clinical samples, and the resources available.

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Austrian, R., 1976. The quellung reaction, a neglected microbiologic technique. Mt Sinai J. Med. 43, 699–709. Batt, S.L., Charalambous, B.M., McHugh, T.D., Martin, S., Gillespie, S.H., 2005. Novel PCRrestriction fragment length polymorphism method for determining serotypes or serogroups of Streptococcus pneumoniae isolates. J. Clin. Microbiol. 43, 2656–2661. Bentley, S.D., Aanensen, D.M., Mavroidi, A., Saunders, D., Rabbinowitsch, E., Collins, M., Donohoe, K., Harris, D., Murphy, L., Quail, M.A., Samuel, G., Skovsted, I.C., Kaltoft, M.S., Barrell, B., Reeves, P.R., Parkhill, J., Spratt, B.G., 2006. Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes. PLoS Genet. 2, e31. Bogaert, D., Veenhoven, R.H., Sluijter, M., Sanders, E.A., De Groot, R., Hermans, P.W., 2004. Colony blot assay: a useful method to detect multiple pneumococcal serotypes within clinical specimens. FEMS Immunol. Med. Microbiol. 41, 259–264. Bratcher, P.E., Kim, K.H., Kang, J.H., Hong, J.Y., Nahm, M.H., 2010. Identification of natural pneumococcal isolates expressing serotype 6D by genetic, biochemical and serological characterization. Microbiology 156, 555–560. Brito, D.A., Ramirez, M., de Lencastre, H., 2003. Serotyping Streptococcus pneumoniae by multiplex PCR. J. Clin. Microbiol. 41, 2378–2384. Bronsdon, M.A., O'Brien, K.L., Facklam, R.R., Whitney, C.G., Schwartz, B., Carlone, G.M., 2004. Immunoblot method to detect Streptococcus pneumoniae and identify multiple serotypes from nasopharyngeal secretions. J. Clin. Microbiol. 42, 1596–1600. Brueggemann, A.B., Griffiths, D.T., Meats, E., Peto, T., Crook, D.W., Spratt, B.G., 2003. Clonal relationships between invasive and carriage Streptococcus pneumoniae and serotype- and clone-specific differences in invasive disease potential. J. Infect. Dis. 187, 1424–1432. Calix, J.J., Nahm, M.H., 2010. A new pneumococcal serotype, 11E, has a variably inactivated wcjE gene. J. Infect. Dis. 202, 29–38. Calix, J.J., Nahm, M.H., Zartler, E.R., 2011. Elucidation of structural and antigenic properties of pneumococcal serotype 11A, 11B, 11C, and 11F polysaccharide capsules. J. Bacteriol. 193, 5271–5278. Calix, J.J., Porambo, R.J., Brady, A.M., Larson, T.R., Yother, J., Abeygunwardana, C., Nahm, M.H., 2012. Biochemical, genetic and serological characterization of two capsule subtypes among streptococcus pneumoniae serotype 20 strains: discovery of a new pneumococcal serotype. J. Biol. Chem. Carvalho Mda, G., Pimenta, F.C., Moura, I., Roundtree, A., Gertz Jr., R.E., Li, Z., Jagero, G., Bigogo, G., Junghae, M., Conklin, L., Feikin, D.R., Breiman, R.F., Whitney, C.G., Beall, B.W., 2013. Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract. Peer J. 1, e97. Carvalho, M.D., Jagero, G., Bigogo, G.M., Junghae, M., Pimenta, F.C., Moura, I., Roundtree, A., Li, Z., Conklin, L., Feikin, D.R., Breiman, R.F., Whitney, C.G., Beall, B., 2012. Potential non-pneumococcal confounding of PCR-based determination of serotype in carriage. J. Clin. Microbiol. CDC, C. PCR Deduction of Pneumococcal Serotypes. [Online]. Available:, http://www.cdc. gov/ncidod/biotech/strep/pcr.htm (Accessed 8th July 2011). Chewapreecha, C., Harris, S.R., Croucher, N.J., Turner, C., Marttinen, P., Cheng, L., Pessia, A., Aanensen, D.M., Mather, A.E., Page, A.J., Salter, S.J., Harris, D., Nosten, F., Goldblatt, D., Corander, J., Parkhill, J., Turner, P., Bentley, S.D., 2014. Dense genomic sampling identifies highways of pneumococcal recombination. Nat. Genet. 46, 305–309. Christensen, P., Kahlmeter, G., Jonsson, S., Kronvall, G., 1973. New method for the serological grouping of Streptococci with specific antibodies adsorbed to protein Acontaining staphylococci. Infect. Immun. 7, 881–885. Coonrod, J.D., Rylko, B., 1976. Latex agglutination in the diagnosis of pneumococcal infection. J. Clin. Microbiol. 4, 168–174.

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from clinical samples without the need for culture, which allows for surveillance of vaccine efficacy. However, such methods are probably only appropriate for reference laboratories or research laboratories where large numbers of samples are passed routinely. Capsule-specific gene based methods are rapid, less laborious and cheaper than phenotypic methods. However, they are not able to distinguish between closely related serotypes where gene sequences vary only by few mutations. Although Quellung and Pneumotest-Latex are the most commonly used phenotypic methods, mPCR developed by the CDC in USA is widely used due to its simplicity and ability to produce results rapidly. Similar to the methods described above, mPCR enables the pneumococcal batches to be screened promptly in multiplex manner and facilitates the identification of pneumococcal infection and capsular type simultaneously, and directly from clinical samples without the need for culture. However, the high sequence homology within the capsule-specific gene regions in serotypes sharing the same serogroup and cross-reactivity with closely related non-pneumococcal polysaccharide genes, especially in respiratory samples (Carvalho et al., 2012; Carvalho Mda et al., 2013), are obstacles for the design of serotype-specific primers. Other less common serotyping methods such as immunoblot assays, ELISA and EIA, flow cytometry, PCR-RFLP, FAF-mPCR, PCR/ESI-MS, mPCR/RLB, RT-PCR, microarray, sequetyping and serotyping from whole genome sequences, have been developed and improved through technological advances, but they are rarely used and not within the reach of many laboratories. However, one should be cautious of the error prone nature of certain methods such as PCR-RFLP and sequetyping as they do not employ serotype-specific genes, and therefore could lead to misidentification of pneumococcal samples. As whole genome sequence data from more pneumococcal isolates becomes available, sequence based inference could be utilised to confirm capsular type alongside other genomic analyses, such as the recent detailed genetic characterisation of pre-vaccine pneumococcal isolates in Malawi (Everett et al., 2012) and pneumococcal capsular evolution (Croucher et al., 2011). Other WGS studies confirmed the serotypeswitching events and were able to get more detailed information on the mechanism of these switch events (Golubchik et al., 2012; Wyres et al., 2013; Croucher et al., 2013). Tracking recombination and population evolution events from a larger pneumococcal library showed that in carriage the most recombinant DNA fragments were present in non-typable pneumococci (Chewapreecha et al., 2014). Furthermore, investigation of approximately 200 isolates of PMEN1 (Croucher et al., 2009) and PMEN14 isolates (Mostowy et al., 2014) provided information on when the first multidrug clone had emerged and identified the majority of recombination events that took place within the pneumococcal capsular region. Advances in sequencing and bioinformatics are allowing increasingly large pneumococcal collections (such as the 20,000 pneumococcal genome project) to be sequenced, further enabling high resolution global analysis of pre- and post-vaccine pneumococcal epidemiology (Klugman, 2013). These advances will allow better description not only of serotype and genotype (e.g., MLST, MLVA), but also a detailed description of surface proteins and their allelic variants, which will help inform new pneumococcal vaccine development through reverse vaccinology.

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Application of a target enrichment-based next-generation sequencing protocol for identification and sequence-based prediction of pneumococcal serotypes. BMC Microbiol. 14, 60. Lund, E., Rasmussen, P., 1966. Omni-serum. A diagnostic Pneumococcus serum, reacting with the 82 known types of Pneumococcus. Acta Pathol. Microbiol. Scand. 68, 458–460. Marttinen, P., Hanage, W.P., Croucher, N.J., Connor, T.R., Harris, S.R., Bentley, S.D., Corander, J., 2012. Detection of recombination events in bacterial genomes from large population samples. Nucleic Acids Res. 40, e6. Massire, C., Gertz Jr., R.E., Svoboda, P., Levert, K., Reed, M.S., Pohl, J., Kreft, R., Li, F., White, N., Ranken, R., Blyn, L.B., Ecker, D.J., Sampath, R., Beall, B., 2012. Concurrent serotyping and genotyping of pneumococci by use of PCR and electrospray ionization mass spectrometry. J. Clin. Microbiol. 50, 2018–2025. Mavroidi, A., Aanensen, D.M., Godoy, D., Skovsted, I.C., Kaltoft, M.S., Reeves, P.R., Bentley, S.D., Spratt, B.G., 2007. Genetic relatedness of the Streptococcus pneumoniae capsular biosynthetic loci. J. Bacteriol. 189, 7841–7855. Miernyk, K., Debyle, C., Harker-Jones, M., Hummel, K.B., Hennessy, T., Wenger, J., Rudolph, K., 2011. Serotyping of Streptococcus pneumoniae isolates from nasopharyngeal samples: use of an algorithm combining microbiologic, serologic, and sequential multiplex PCR techniques. J. Clin. Microbiol. 49, 3209–3214. Miller, E., Andrews, N.J., Waight, P.A., Slack, M.P., George, R.C., 2011. Herd immunity and serotype replacement 4 years after seven-valent pneumococcal conjugate vaccination in England and Wales: an observational cohort study. Lancet Infect. Dis. 11, 760–768. Morais, L., Carvalho Mda, G., Roca, A., Flannery, B., Mandomando, I., Soriano-Gabarro, M., Sigauque, B., Alonso, P., Beall, B., 2007. 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