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Research Article| Volume 46, P100-106, May 2016

Serotypes and genotypes of Streptococcus pneumoniae isolates from Trinidad and Tobago

Open AccessPublished:April 07, 2016DOI:https://doi.org/10.1016/j.ijid.2016.04.005
Serotypes and genotypes of Streptococcus pneumoniae isolates from Trinidad and Tobago
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      Highlights

      • •
        Most Streptococcus pneumoniae isolates were from blood, children under 2 years old, and were recovered after the introduction of conjugate vaccines.
      • •
        Major serotypes detected were 19F, 6B, 23F and 3.
      • •
        Fifty-six different sequence types (STs) were identified; predominant STs were ST138, ST145, ST8398, ST10440, ST320.
      • •
        Some isolates were identified as being related to seven of the internationally described Pneumococcal Molecular Epidemiology Network (PMEN) clones.
      • •
        The prevailing pneumococcal genotypes in the country reveals that both PCV10 and PCV13 provide good coverage.

      Summary

      Objectives

      There are currently 94 known pneumococcal capsular polysaccharide serotypes and their prevalence differs by geographic region and the period studied. Streptococcus pneumoniae infections have been diagnosed clinically in Trinidad and Tobago and other Caribbean countries, however data on the serotype and sequence type distributions in this country are limited. The objective of this study was to determine serotypes and multilocus sequence types (MLSTs) of invasive and non-invasive pneumococcal isolates from Trinidad and Tobago.

      Methods

      Ninety-eight pneumococcal isolates from several regional hospitals in the country were analyzed using both standard microbiological methods and molecular analysis. These isolates included invasive (n = 83) and selected non-invasive (n = 15) strains recovered before (n = 25) and after (n = 73) the introduction of the pneumococcal conjugate vaccine.

      Results

      More than half of the isolates (54.1%) were recovered from children under 15 years of age, with the largest proportion being from children under 2 years of age (24.5%). The most prevalent serotypes were 19F (18.4%), 6B (15.3%), 23F (14.3%), 3 (11.2%), 19A (6.1%), 6A (5.1%), 14 (5.1%), and 9V (4.1%). The most common serotype/MLST combinations were 6B/ST138 (n = 10, 10.2%), 3/ST180 (n = 5, 5.1%), 23F/ST629 (n = 5, 5.1%), 19F/ST8398 (n = 4, 4.1%), and three each of 6B/ST145, 14/9V/ST156, 9V/ST162, 19A/320, and 3/ST10440.

      Conclusions

      This report provides the first glimpse of the prevailing pneumococcal sequence types in the country. Most of the isolates represented serotypes in the 10-valent (61.2% of isolates) and 13-valent (83.7%) pneumococcal conjugate vaccines. A detailed population study is warranted to fully determine the circulating pneumococcal sequence types. Furthermore, the implementation of an effective and continuous surveillance system in Trinidad and Tobago is paramount to monitor vaccine impact.

      Keywords

      1. Introduction

      Streptococcus pneumoniae continues to be a major cause of morbidity and mortality in humans.
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      Each serotype is distinguished based on the chemical composition of and antigenic differences in the capsular polysaccharide.
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      PPV23 is recommended for use in the elderly and immune-compromised individuals over the age of 2 years, while the PCVs are recommended for children under 2 years and also for older children. In late 2011, PCV13 was also recommended for adults ≥50 years who are clinically at risk, including adults over 65 years of age.
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      J Korean Med Sci. 2013; 28: 4-15
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      Pediatric invasive pneumococcal disease in the United States in the era of pneumococcal conjugate vaccines.
      Clin Microbiol Rev. 2012; 25: 409-419
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      Pneumococcal conjugate vaccine for childhood immunization—WHO position paper.
      Wkly Epidemiol Rec. 2012; 87: 129-144
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      Advisory Committee on Immunization Practices licensure of a 13-valent pneumococcal conjugate vaccine (PCV13) and recommendations for use among children. MMWR Morb Mortal Wkly Rep 2010;59:258–61.

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      PCV7 was formulated to include the most frequent serotypes causing pediatric IPD at that time in developed countries, in particular the USA (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F).
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      Wkly Epidemiol Rec. 2012; 87: 129-144
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      Two PCVs are currently available for use in children, PCV10 (PCV7 serotypes plus 1, 5, and 7F; PHiD-CV, Synflorix, GlaxoSmithKline) and PCV13 (PCV7 serotypes plus 1, 3, 5, 6A, 7F, and 19A; Prevnar, Pfizer); these replaced PCV7.
      • Tan T.
      Pediatric invasive pneumococcal disease in the United States in the era of pneumococcal conjugate vaccines.
      Clin Microbiol Rev. 2012; 25: 409-419
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      Wkly Epidemiol Rec. 2012; 87: 129-144
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      Advisory Committee on Immunization Practices licensure of a 13-valent pneumococcal conjugate vaccine (PCV13) and recommendations for use among children. MMWR Morb Mortal Wkly Rep 2010;59:258–61.

      , 
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      MMWR. 2014; 63: 822-825
      The current vaccines provide wider serotype coverage than the previous PCV7 formulation.
      • Song J.Y.
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      Clinical implications of pneumococcal serotypes: invasive disease potential, clinical presentations, and antibiotic resistance.
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      In the Caribbean, as well as other regions, vaccine uptake has been variable despite its approval for use. Although PCV7 was introduced in the USA in 2000, it was not until March 2010 that it was introduced into the national immunization program (NIP) in Trinidad and Tobago for infants at risk of pneumococcal disease, mainly those with immune deficiencies and other chronic diseases. Prior to 2010, PCV7 was only available in the private sector for certain children with medical conditions that put them at higher risk of disease.

      Caribbean Epidemiology Centre annual report 2008. CAREC/PAHO/WHO. Published 2009. Available at: http://carpha.org/downloads/200820CAREC20Annual20Report.pdf (accessed March 5, 2015).

      In 2011, the PCV10 vaccine was introduced, followed by its widespread use in the NIP for all children aged <2 years.

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      Caribbean Epidemiology Centre annual report 2010. CAREC/PAHO/WHO. Published 2011. Available at: http://carpha.org/downloads/201020CAREC20Annual20Report.pdf (accessed March 5, 2015).

      Subsequently, PCV13 was introduced into the NIP in August 2015, replacing PCV10. Vaccine coverage with PCV10 was reported to be 95% as of December 2014.

      World Health Organization. Vaccine-preventable diseases: monitoring system. 2014 global summary. Published online 2014 (Updated June 2015) WHO. Available at: http://apps.who.int/immunization_monitoring/globalsummary (accessed February 27, 2016).

      PPV23 was licensed in Trinidad and Tobago in 2004, but was only incorporated into the NIP in 2012 for children over 2 years of age with chronic diseases, adults 65 years and above, and persons at risk of pneumococcal disease, including those with HIV/AIDS.

      Caribbean Epidemiology Centre annual report 2010. CAREC/PAHO/WHO. Published 2011. Available at: http://carpha.org/downloads/201020CAREC20Annual20Report.pdf (accessed March 5, 2015).

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      World Health Organization. Vaccine-preventable diseases: monitoring system. 2014 global summary. Published online 2014 (Updated June 2015) WHO. Available at: http://apps.who.int/immunization_monitoring/globalsummary (accessed February 27, 2016).

      Trinidad and Tobago does not qualify for donor support under the Global Alliance for Vaccines and Immunizations (GAVI) program.

      GAVI Alliance. Countries approved for support March 2015. Geneva, Switzerland: GAVI Alliance; Published June 2015. Available at: http://www.gavialliance.org/results/countries-approved-for-support (accessed August 8, 2014).

      However, vaccines are provided by the Trinidad and Tobago Government under their immunization program and are purchased through a Pan American Health Organization (PAHO) revolving fund.

      Caribbean Epidemiology Centre annual report 2010. CAREC/PAHO/WHO. Published 2011. Available at: http://carpha.org/downloads/201020CAREC20Annual20Report.pdf (accessed March 5, 2015).

      Although pneumococcal infections are clinically diagnosed and S. pneumoniae are routinely isolated from specimens, data on the serotype distribution in the Caribbean, and in Trinidad and Tobago in particular, are limited.

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      The use of molecular methods to determine genotypes of strains in the Caribbean, as in many Latin American countries, has been limited due to financial constraints and other logistical difficulties.
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      To the best of the authors’ knowledge, there is no published study that has used multilocus sequence typing (MLST) to characterize pneumococcal isolates from Trinidad and Tobago. The objective of this study was to characterize pneumococcal isolates from both invasive and selected non-invasive sites using serotyping and MLST, and to compare these genotypes to well-described international clonal complexes (http://www.pneumogen net/pmen/).
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      2. Materials and methods

      2.1 Bacterial isolates and country demographics

      All pneumococcal isolates (n = 73) obtained from routine clinical specimens submitted to the microbiology diagnostic laboratories of the five major public hospitals in Trinidad and Tobago, during the period 2011 to 2013, were included in this study. The isolates were included in the analysis if they met the following selection criteria: (1) patients with evidence of ongoing infection or confirmed diagnosis (clinical features including fever, elevated C-reactive protein, and elevated white cell count); (2) S. pneumoniae as the only pathogen isolated from both invasive and non-invasive sites. Duplicate isolates from the same episode of infection in any one patient were considered to represent the same episode, and therefore were counted once. Selected isolates from non-invasive sites were also included to ascertain a comprehensive picture of the molecular characteristics of pneumococcal infections.
      A number of historical pneumococcal isolates (n = 25) from clinical specimens that were collected between 1997 and 2010 were also included in the analysis. Most of the strains were collected from three regional hospitals, prior to the commencement of the SIREVA (Sistema Regional de Vacinas), the Regional System for Vaccines in the Americas project in Trinidad and Tobago.

      Caribbean Epidemiology Centre annual report 2008. CAREC/PAHO/WHO. Published 2009. Available at: http://carpha.org/downloads/200820CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 
      • Di Fabio J.C.
      • Castaneda E.
      • Leal L.
      • Castillo O.
      PAHO SIREVA-VIGIA Study Group
      Evolution of Streptococcus pneumoniae serotypes and penicillin susceptibility in Latin America, Sireva-Vigia Group, 1993–1999.
      Pediatr Infect Dis J. 2001; 20: 259-267
      , 
      • Garcia S.
      • Levine O.S.
      • Cherian T.
      • Gabastou J.M.
      • Andrus J.
      Working Group members
      Pneumococcal disease and vaccination in the Americas: an agenda for accelerated vaccine introduction.
      Rev Panam Salud Publica. 2006; 19: 340-348
      These historical isolates were included to give some idea of the serotypes and sequence types present in the country prior to the introduction of the PCV7 in the vaccination program in Trinidad and Tobago. The isolates from the earlier period (1997–1998) were not part of a specific study. The pre- and post-vaccination periods were defined as the years 1997 to February 2010 and March 2010 to 2013, respectively.
      Demographic and clinical parameters were obtained using a standardized questionnaire. Conventional phenotypic identification was performed at the microbiology laboratories of the regional hospitals in the country. The twin islands of Trinidad and Tobago are the most southern islands in the Caribbean, located just seven miles off the north eastern coast of Venezuela, and have an estimated population of 1.3 million. It is estimated that 19.4% of the population are less than 14 years old and 9.5% are 65 years old and above. About 13.7% of the population live in urban areas, with 66 000 people in the major capital city of Port of Spain. The majority (68.6%) of the population comprises people of African and Indian descent (34.2% and 34.4%, respectively), with people of mixed race, European, Chinese, and Middle Eastern ancestry adding diversity to the ethnic mix.

      Central Intelligence Agency (CIA). The World Fact Book. Trinidad and Tobago. People and Society. 2015. Available at https://www.cia.gov/library/publications/resources/the-world-factbook/geos/td.html (accessed February 2015).

      All confirmed pneumococcal strains were collected and stored in brain–heart infusion broth with glycerol at −70 °C until further conventional and molecular analysis.
      Ethical approval was obtained from the Ethics Committee of the University of the West Indies at St. Augustine. Permission was also granted by the ethics committees of all the regional hospitals where the studies were carried out, namely the North West Regional Health Authority, North Central Regional Health Authority, Eastern Regional Health Authority, South West Regional Health Authority, and Scarborough General Hospital Tobago. Patient records and information were anonymized and de-identified prior to analysis.

      2.2 Capsular serotyping

      All isolates were serotyped by conventional Quellung reaction method at the US Centers for Disease Control and Prevention (CDC) Streptococcus Laboratory (CDC, Atlanta, GA, USA). Further serotyping was performed using a sequential multiplex PCR method, as described by Pai et al., and updated on the CDC Streptococcus Laboratory website.
      • Pai R.
      • Gertz R.
      • Beall B.
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      J Clin Microbiol. 2006; 44: 124-131
      , 

      Centers for Disease Control and Prevention. Multiplex PCR for deducing serotypes from pneumococcal isolates/PCR serotype deduction protocols/PCR deduction of pneumococcal serotypes. Atlanta, GA: CDC; 2014. Available at: http://www.cdc.gov/ncidod/biotech/strep/pcr.htm (accessed March 23, 2015).

      Genomic DNA was extracted from the bacterial isolates using Chelex 100 (Bio-Rad Laboratories, Hercules, CA, USA).

      2.3 Multilocus sequence typing analysis

      MLST was performed as described by Enright and Spratt, with modified primers (http://www.cdc.gov/streplab/alt-mlst-primers.html).
      • Enright M.C.
      • Spratt B.G.
      A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease.
      Microbiology. 1998; 144: 3049-3060
      Briefly, the internal fragments of seven housekeeping genes (aroE, gdh, gki, recP, spi, xpt, and ddl) of the pneumococcal genome were amplified and sequenced, then the sequence types (STs) were determined by comparing the sequences with alleles from the pneumococcal MLST database (http://pubmlst.org/spneumoniae/). New alleles and STs were submitted to the database curator for assignment. Clonal complexes were defined as groups of STs sharing ≥5 alleles with one or more members of the set and were assigned using eBURST version 3 program (http://eburst.mlst.net) and arbitrarily assigned CC1–CC13.

      3. Results

      3.1 Bacterial isolates

      Ninety-eight S. pneumoniae isolates were obtained; 83 were invasive isolates (from blood, pleural fluid, cerebrospinal fluid, and swab from brain tissue) and 15 were non-invasive isolates (ear, eye swabs, and sputum). Most of the invasive isolates were from blood (86.7%, 72/83), while the majority of the non-invasive isolates were from ear swabs (73.3%, 11/15). More than half of the isolates (54.1%, 53/98) were recovered from children under 15 years of age (Table 1), with the largest proportion being from children under 2 years of age (24.5%, 24/98). Twenty-five (25.5%) pneumococcal isolates were recovered before the introduction of the conjugate vaccines (1997 to February 2010) and 73 (74.4%) after (March 2010 to 2013).
      Table 1Age and serotype distribution of invasive and non-invasive pneumococcal isolates from Trinidad and Tobago
      Age (years)No. of isolates (%)Serotype (No. of isolates)
      <224 (24.5%)6B (6), 9V (2), 14 (1), 19F (8), 23F (2), 3 (1), 10A (2), 16F (1), 22F (1)
      2 to <518 (18.4%)6B (4), 19F (3), 23F (5) 6A (2), 19A (3), 15C (1)
      5 to <1511 (11.2%)14 (1), 19F (2), 23F (3), 3 (1), 6A (1), 15C (1), 18C (1)
      15 to <241 (1.0%)11A (1)
      24 to <4515 (15.3%)9V (2), 6B (1), 14 (2), 19F (3), 23F (1), 3 (1), 19A (1), 11A (1), 15A (1), 17F (1), 35F (1)
      45 to <6011 (11.2%)6B (2), 14 (1), 23F (2), 7F (1), 3 (2), 6A (1), 19F (1), 20 (1), 11A (1)
      >6018 (18.4%)4 (1), 6B (2), 18C (1), 19F (1), 23F (1), 3 (6), 6A (1), 19A (2), 9N (1), 15B (1), 15A (1)

      3.2 Serotype distribution

      Twenty-two different serotypes were detected among the 98 isolates. Only eight serogroups/serotypes were represented by at least four or more isolates (Table 1) and these accounted for 74.4% of the serotypes. These major serotypes included the following: 19F (18.4%), 6B (15.3%), 23F (14.2%), 3 (11.2%), 19A (6.1%), 6A (5.1%), 14 (5.1%), and 9V (4.1%). For the historical isolates (pre-PCV7 period, 1997–2010), the most prevalent serotypes in descending order were 19F (n = 7), 3 (n = 5), 6B (n = 4), 9V (n = 4), and 23F (n = 2); 7.1% were non-vaccine types (NVT). In the post-PCV7 period (March 2010 to 2013), serotypes 19F, 23F, 6B, 3, 19A, and 6A were the most common, and 32.6% were NVT. Six serotypes (3, 6B, 11A, 14, 19F, and 23F) were identified in both periods. The total coverage rates of PCV7, PCV10, and PCV13 were 60.2%, 61.2%, and 83.7%, respectively; there was a significant 22.5% difference between PCV10 and PCV13 in coverage.

      3.3 Clonal structure

      The 98 pneumococcal isolates included in this study represented 55 different STs (Table 2). Nine STs accounted for more than 39.7% of the isolates, including ST138 (n = 10), ST180 (n = 5), ST629 (n = 5), ST8398 (n = 4), and ST145, ST156, ST162, ST320, and ST10440 (each accounting for three isolates). Fifteen (15.3%) new STs, including six new MLST alleles, were identified in this study and were assigned to ST 9743–9751, 10439, 10440, 10446, 10449, 10450, and 10455 (Table 2). When compared with STs that have previously been deposited in the MLST database, eight of the 15 were related to previously reported STs. Of these 15 new STs, eight were identified during the period 1997–2009, including ST10440, which was also found during the post-vaccination period (2011–2013).
      Table 2Distribution of genotype (ST) among the pneumococcal serotypes from Trinidad and Tobago, 1997–2013
      SerotypeNo. of isolatesSequence type (No. of isolates)MLST_aroe-gdh-gki-recp-spi-xpt-ddlClonal complex
      4120510-5-4-5-13-10-18Singleton
      6B15138 (8)

      145 (3)

      146 (2)

      497 (1)

      4241 (1)      		7-5-8-5-10-6-14

      7-6-1-1-6-15-14

      7-6-1-2-6-15-14

      7-25-4-2-48-20-28

      15-16-19-10-6-20-26      		Singleton

      5

      5

      12

      Singleton
      9V4156 (1)

      162 (3)      		7-11-10-1-6-8-1

      7-11-10-1-6-8-14      		2

      2
      145156 (2)

      554 (2)

      10449 (1)      		7-11-10-1-6-8-1

      8-8-4-15-39-12-26

      7-11-10-1-451-8-1      		2

      Singleton

      2
      18C249642-35-29-36-9-39-1813
      19F18138
      These serotype 19F isolates are wzy variants described by Pimenta et al.48
      (1)

      236 (1)

      425 (1)

      485 (2)

      4251 (1)

      793 (1)

      3907 (1)

      6937 (1)

      9743 (1)

      9744 (1)

      9746 (1)

      9748 (1)

      8398
      These serotype 19F isolates are wzy variants described by Pimenta et al.48
      (4)

      10450 (1)      		7-5-8-5-10-6-14

      15-16-19-15-6-20-26

      1-5-1-5-1-1-8

      1-5-1-1-1-1-8

      15-16-19-10-6-20-26

      7-13-9-1-6-19-14

      1-5-214-12-1-79-8

      2-5-4-5-27-20-5

      1-5-106-12-10-4-14

      15-16-19-15-6-4-26

      18-43-4-1-6-4-8

      18-5-4-1-6-11-8

      7-11-10-1-8-8-1

      327-5-4-5-27-20-5      		Singleton

      3

      6

      6

      3

      11

      Singleton

      9

      Singleton

      3

      10

      10

      2

      9
      23F1436 (2)

      37 (1)

      439 (1)

      629 (5)

      9747 (1)

      9749 (1)

      9751 (1)

      10446 (2)      		1-8-4-1-1-4-6

      1-8-6-2-6-4-6

      1-8-9-2-6-4-6

      1-8-9-10-6-4-6

      2-13-2-4-9-1-1

      1-8-9-6-6-4-6

      1-2-9-10-6-4-6

      1-8-9-294-6-4-6      		Singleton

      1

      1

      1

      4

      1

      1

      1
      7F11918-9-2-1-6-1-17Singleton
      311180 (5)

      1116 (1)

      662 (1)

      10440 (3)

      10455 (1)      		7-15-2-10-6-1-22

      1-26-28-11-13-1-14

      5-35-29-12-9-39-18

      1-26-28-11-452-1-14

      2-12-19-1-6-615-22      		Singleton

      14

      13

      14

      Singleton
      6A5473 (2)

      138 (1)

      460 (1)

      490 (1)      		7-25-4-4-15-20-28

      7-5-8-5-10-6-14

      5-7-4-10-10-1-27

      2-13-9-1-6-19-14      		12

      Singleton

      7

      11
      19A



      9N      		6



      1      		320 (3)

      695 (2)

      199 (1)

      66      		4-16-19-15-6-20-1

      16-13-4-4-6-113-18

      8-13-14-4-17-4-14

      2-8-2-4-6-1-1      		3

      Singleton

      Singleton

      4
      10A247535-241-4-2-10-1-277
      11A362 (2)

      9750 (1)      		2-5-29-12-16-3-14

      2-5-29-12-16-1-14      		8

      8
      15A2732-13-2-15-6-1-14
      15B11998-13-14-4-17-4-14Singleton
      15C212627-41-2-6-10-26-1Singleton
      16F1104391-10-62-10-15-1-31Singleton
      17F13927-5-1-1-6-31-145
      20197455-31-8-295-9-1-665Singleton
      22F14331-1-4-1-18-58-17Singleton
      35F14982-7-4-16-10-40-27Singleton
      STs underlined refer to new STs identified in this study. New alleles in this study are in bold.
      a These serotype 19F isolates are wzy variants described by Pimenta et al.48
      STs that represented more than one serotype included ST138 (6A, 6B, 19F), ST156 (9V, 14), and ST199 (15B, 19A). Twelve serotypes (4, 7F, 9N, 10A, 15A, 15B, 15C, 16F, 17F, 18C, 22F, 35F) were associated with a single ST, whereas the remaining serotypes consisted of multiple STs. Serotypes 19F and 23F showed the most genetic diversity with 14 and eight STs, respectively. Four STs were isolated during both the pre- and post-PCV periods: STs 62, 156, 180, and 10440. The eBURST analysis revealed 14 clonal complexes (CC) and 17 singletons containing 63 and 35 isolates, respectively (Table 2). Of the new STs, five were singletons and 10 grouped into CCs. Fourteen isolates (14.3%) were identified as being related to seven of the internationally described Pneumococcal Molecular Epidemiology Network (PMEN) clones (PMEN3, PMEN4, PMEN14, PMEN31, PMEN37, PMEN38, and PMEN39; http://www.pneumogen.net/pmen/) (Figure 1).
      • McGee L.
      • McDougal L.
      • Zhou J.
      • Spratt B.G.
      • Tenover F.C.
      • George R.
      • et al.
      Nomenclature of major antimicrobial-resistant clones of Streptococcus pneumoniae defined by the pneumococcal molecular epidemiology network.
      J Clin Microbiol. 2001; 39: 2565-2571
      Figure thumbnail gr1
      Figure 1eBURST diagram showing a population snapshot of 98 isolates from Trinidad and Tobago with complete MLST data. Each ST is represented by a point, with the size of the point representing the number of isolates associated with that ST within the dataset. Isolates (14.3%) were related to seven of the internationally described PMEN clones (http://www.pneumogen.net/pmen/). STs circled represent new STs identified among isolates in this study.

      4. Discussion

      This study aimed to describe the observed prevailing serotypes and genotypes of pneumococcal isolates from both invasive and non-invasive pneumococcal diseases in Trinidad and Tobago. IPD is not a notifiable disease in Trinidad and Tobago, and the current laboratory-based surveillance system is based on voluntary rather than mandatory submission of isolates. The annual burden of pneumococcal disease in Latin America and the Caribbean has been estimated to be 1.6 million in children less than 5 years of age.
      • Constenla D.
      • Gomez E.
      • de la Hoz F.P.
      • O’Loughlin R.
      • Sinha A.
      • Valencia J.E.
      • Valenzuela M.T.
      The burden of pneumococcal disease and cost-effectiveness of a pneumococcal vaccine in Latin America and the Caribbean: a review of the evidence and a preliminary economic analysis.
      The Albert B. Sabin Vaccine Institute, Pan American Health Organization, GAVI's Pneumococcal Accelerated Development Introduction Plan at Johns Hopkins (PneumoADIP), Centers for Disease Control and Prevention, Washington, DC2007
      In Trinidad and Tobago, the incidence rates of fever and acute respiratory infections (ARI) during the years 2009–2010 in ages less than 5 was 22 407–25, 202 cases per 100,000 and for ages over 5 years, it decreased from 2447–1163 cases per 100,000 for the same corresponding years.

      Caribbean Epidemiology Centre annual report 2010. CAREC/PAHO/WHO. Published 2011. Available at: http://carpha.org/downloads/201020CAREC20Annual20Report.pdf (accessed March 5, 2015).

      A number of pneumococcal infections in both children and adult patients were identified during the two time periods, before (1997 to February 2010) and after (March 2010 to 2013) PCV vaccination. A total of 98 isolates were available for analysis, with the majority recovered during the years 2011–2013. There were only 25 isolates available for the period 1997–2010 (21 from 1997–1999 and four from 2007–2010), representing less than 2% of the pneumococcal isolates reported between 2007 and 2010.

      Caribbean Epidemiology Centre annual report 2008. CAREC/PAHO/WHO. Published 2009. Available at: http://carpha.org/downloads/200820CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre annual report 2009. CAREC/PAHO/WHO. Published 2010. Available at: http://carpha.org/downloads/200920CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre annual report 2010. CAREC/PAHO/WHO. Published 2011. Available at: http://carpha.org/downloads/201020CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2007. CAREC/PAHO/WHO. Published 2008. Available online at: http://carpha.org/downloads/2007CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2011. CAREC/PAHO/WHO; Published 2012. Available online at: http://carpha.org/downloads/2011CAREC20Annual20Report.pdf (accessed March 5, 2015).

      The majority of the isolates collected during these periods were not available for the current study analysis due to non-viability after storage. This is unfortunate, because it was a missed opportunity to further characterize samples prior to 2010.
      The coverage rates of PCV7, PCV10, and PCV13 for pneumococcal isolates encountered in this study were 60.2%, 61.2%, and 83.7%, respectively. The observed 22.5% difference in PCV10 and PCV13 coverage rates is noteworthy. PCV10 provided little added benefit to PCV7, as two of the three serotypes (1 and 5) were rare in Trinidad and Tobago,

      Caribbean Epidemiology Centre annual report 2008. CAREC/PAHO/WHO. Published 2009. Available at: http://carpha.org/downloads/200820CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre annual report 2009. CAREC/PAHO/WHO. Published 2010. Available at: http://carpha.org/downloads/200920CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre annual report 2010. CAREC/PAHO/WHO. Published 2011. Available at: http://carpha.org/downloads/201020CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre annual report 2001. CAREC/PAHO/WHO. Published 2002. Available at: http://carpha.org/downloads/200120CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre annual report 2002. CAREC/PAHO/WHO. Published 2003. Available at: http://carpha.org/downloads/200220CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2005. CAREC/PAHO/WHO; Published 2006. Available at: http://carpha.org/downloads/2005CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2006. CAREC/PAHO/WHO. Published 2007. Available online at: http://carpha.org/downloads/2006CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2007. CAREC/PAHO/WHO. Published 2008. Available online at: http://carpha.org/downloads/2007CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2011. CAREC/PAHO/WHO; Published 2012. Available online at: http://carpha.org/downloads/2011CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      PAHO. Informe Regional de SIRVEA11: Datos por país y por grupos de edad sobre las características de los aislamientos de Streptococcus pneumoniae, Haemophilus influenzae y Neisseria meningitidis en procesos invasores 2000–2005. Washington DC: OPS; 2007.

      whereas the additional serotypes in PCV13 (3, 6A, and 19A) contributed to the increase in vaccine coverage that was observed. Data published by the SIREVA network and the Caribbean Public Health Agency (CARPHA), formerly the Caribbean Epidemiology Centre (CAREC), have shown that the most prevalent serogroups/serotypes causing IPD in the Caribbean prior to the NIP in Trinidad and Tobago were 14, 6B, 23F, 18, 19, 19F, 9, 6A, 1, and 23.

      Caribbean Epidemiology Centre annual report 2001. CAREC/PAHO/WHO. Published 2002. Available at: http://carpha.org/downloads/200120CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre annual report 2002. CAREC/PAHO/WHO. Published 2003. Available at: http://carpha.org/downloads/200220CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2005. CAREC/PAHO/WHO; Published 2006. Available at: http://carpha.org/downloads/2005CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2006. CAREC/PAHO/WHO. Published 2007. Available online at: http://carpha.org/downloads/2006CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2007. CAREC/PAHO/WHO. Published 2008. Available online at: http://carpha.org/downloads/2007CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      PAHO. Informe Regional de SIRVEA11: Datos por país y por grupos de edad sobre las características de los aislamientos de Streptococcus pneumoniae, Haemophilus influenzae y Neisseria meningitidis en procesos invasores 2000–2005. Washington DC: OPS; 2007.

      Subsequently, serogroups 19 and 9 were identified as 19F and 9V, respectively, by the Instituto Nacional de Salud Bogotá, Bogotá, Colombia.

      PAHO. Informe Regional de SIRVEA11: Datos por país y por grupos de edad sobre las características de los aislamientos de Streptococcus pneumoniae, Haemophilus influenzae y Neisseria meningitidis en procesos invasores 2000–2005. Washington DC: OPS; 2007.

      During the same period in Trinidad and Tobago, 12 different serogroups and 22 serotypes were identified, the most prevalent being 14, 6B, 4, 6A, and 15C. Serotypes 14, 6B, 23F, and 4 were recovered from children under 5 years of age.

      Caribbean Epidemiology Centre annual report 2001. CAREC/PAHO/WHO. Published 2002. Available at: http://carpha.org/downloads/200120CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre annual report 2002. CAREC/PAHO/WHO. Published 2003. Available at: http://carpha.org/downloads/200220CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2005. CAREC/PAHO/WHO; Published 2006. Available at: http://carpha.org/downloads/2005CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2006. CAREC/PAHO/WHO. Published 2007. Available online at: http://carpha.org/downloads/2006CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2007. CAREC/PAHO/WHO. Published 2008. Available online at: http://carpha.org/downloads/2007CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 
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      Pneumococcal infections in Trinidad: patterns of antimicrobial susceptibility: 1994–2002.
      Japan J Infect Dis. 2005; 58: 20-24
      When compared globally to those observed in Latin America, North America, and Europe, the 10 prevalent serotypes prior to 2010 in the present study were comparably well represented in samples from elsewhere in the pre-PCV7 and early post-PCV7 eras.
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      , 

      PAHO. Informe Regional de SIRVEA11: Datos por país y por grupos de edad sobre las características de los aislamientos de Streptococcus pneumoniae, Haemophilus influenzae y Neisseria meningitidis en procesos invasores 2000–2005. Washington DC: OPS; 2007.

      , 
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      • Castillo O.
      PAHO SIREVA-VIGIA Study Group
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      Pediatr Infect Dis J. 2001; 20: 259-267
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      Serotypes 1 and 5 were absent during the period 1997–2013 in the present analysis. This is similar to North America, where serotypes 1 and 5 are uncommon, but is different from the situation in some European, Asian, and Latin American countries, where they are frequently isolated.
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      PLoS Med. 2010; 7: e1000348
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      • Siber G.R.
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      Clin Infect Dis. 2000; 30: 100-121
      , 
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      • Castaneda E.
      • Leal L.
      • Castillo O.
      PAHO SIREVA-VIGIA Study Group
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      Pediatr Infect Dis J. 2001; 20: 259-267
      , 
      • Castaneda E.
      • Leal L.
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      Pneumococcal Study Group in Colombia
      Distribution of capsular types and antimicrobial susceptibility of invasive isolates of Streptococcus pneumoniae in Colombian children.
      Microbiol Drug Resist. 1997; 3: 147-152
      , 
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      Streptococcus pneumoniae serotype 19A in Latin America and the Caribbean: a systematic review and meta-analysis, 1990–2010.
      BMC Infect Dis. 2012; 12: 124
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      Pneumococci causing invasive disease in children prior to the introduction of pneumococcal conjugate vaccine in Scotland.
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      Serotype 6C, which has been identified in some countries,
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      was absent in the isolates tested in the present study. It is unclear whether 6C may have been among the serogroup 6A/6B isolates reported by CAREC from 1997 to 1999 and 2007 to 2010, especially among those isolated after the introduction of PCV7.

      Caribbean Epidemiology Centre annual report 2008. CAREC/PAHO/WHO. Published 2009. Available at: http://carpha.org/downloads/200820CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre annual report 2009. CAREC/PAHO/WHO. Published 2010. Available at: http://carpha.org/downloads/200920CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre annual report 2010. CAREC/PAHO/WHO. Published 2011. Available at: http://carpha.org/downloads/201020CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre annual report 2001. CAREC/PAHO/WHO. Published 2002. Available at: http://carpha.org/downloads/200120CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre annual report 2002. CAREC/PAHO/WHO. Published 2003. Available at: http://carpha.org/downloads/200220CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2005. CAREC/PAHO/WHO; Published 2006. Available at: http://carpha.org/downloads/2005CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2006. CAREC/PAHO/WHO. Published 2007. Available online at: http://carpha.org/downloads/2006CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2007. CAREC/PAHO/WHO. Published 2008. Available online at: http://carpha.org/downloads/2007CAREC20Annual20Report.pdf (accessed March 5, 2015).

      , 

      Caribbean Epidemiology Centre Annual Report 2011. CAREC/PAHO/WHO; Published 2012. Available online at: http://carpha.org/downloads/2011CAREC20Annual20Report.pdf (accessed March 5, 2015).

      Studies in the USA have shown that previously typed 6A isolates recovered after PCV7 introduction were primarily 6C.
      • Carvalho M.D.
      • Pimenta F.C.
      • Gertz R.E.
      • Joshi H.H.
      • Trujillo A.A.
      • Keys L.E.
      • et al.
      PCR-based quantitation and clonal diversity of the current prevalent invasive serogroup 6 pneumococcal serotype, 6c, in the United States in 1999 and 2006 to 2007.
      J Clin Microbiol. 2009; 47: 554-559
      Serotype 6C was first described in 2006, however retrospective analyses have indicated that it was in existence as early as 1962. Prior to PCV7, 6C was extremely rare in children <5 years old and was uncommon in adults.
      • Carvalho M.D.
      • Pimenta F.C.
      • Gertz R.E.
      • Joshi H.H.
      • Trujillo A.A.
      • Keys L.E.
      • et al.
      PCR-based quantitation and clonal diversity of the current prevalent invasive serogroup 6 pneumococcal serotype, 6c, in the United States in 1999 and 2006 to 2007.
      J Clin Microbiol. 2009; 47: 554-559
      , 
      • McEllistrem C.M.
      • Nahm M.H.
      • Novel C.M.
      Novel pneumococcal serotypes 6C and 6D: anomaly or harbinger.
      Clin Infect Dis. 2012; 55: 1379-1386
      , 
      • Green M.C.
      • Mason E.O.
      • Kaplan S.L.
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      • Stovall S.H.
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      and there were several examples of capsular switching events in this study: ST138 (6A, 6B, 19F), ST156 (9V, 14), and ST199 (15B, 19A). Eight STs (138, 180, 629, 8398, 145, 156, 162, and 10440) accounted for more than 36% of the isolates, and a number of isolates (14.3%) belonged to well-described international PMEN clones (http://www.pneumogen.net/pmen/). Of the 39 STs reported, 10 from Latin American countries and 29 from North America have been deposited previously in the MLST database (http://pubmlst.org/spneumoniae/). Some of these STs have significant geographic spread, for example ST138 and ST180 have been found to be present in most regions, including the USA and some European and Latin American countries.
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      and Germany (described in the MLST database). Available data on genotypes from Venezuela are limited, and the MLST database has 29 isolates deposited since 2007. Among these isolates, only three STs (ST320, ST156, and ST199) were similar to those from the Trinidad and Tobago study. Four STs were observed in both the pre- and post-vaccination periods (1997 to February 2010 and March 2010 to 2013): ST62, 156, 180, and 10440. ST62 (11A) was isolated both in 1998 and 2012, and this ST has been described in invasive isolates from Spain and Italy (described in the MLST database).
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      When the clonal composition was compared with the USA and Scotland in the pre- and the early post-vaccination periods, variations were observed. For instance, serotype 14 was represented by four STs in the USA and 12 STs in Scotland during the pre-PCV era, and quite striking was the absence of ST156 in Scotland.
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      ST156 has frequently occurred worldwide and was first described as serotype 9V; this has subsequently also been described as serotype 14 and as other serotypes.
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      Furthermore, ST156 is often associated with antibiotic resistance and is one of the well described PMEN clones (Spain9V-ST156, PMEN3) that have spread globally, including within the Latin America region.
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      Isolates belonging to clonal complex 156 (CC156) were also found to modestly emerge within 19A in the post-PCV period in the USA.
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      It is also interesting to see this very enduring clonal complex associated with several serotypes in the post-PCV13 era.
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      In the present study, ST156 was associated with both serotype 9V and 14 (commonly found in South America), and CC156 included closely related ST8398 (19F), ST162 (9V), and ST10449 (14). PMEN4 (Tennessee23F-ST37) was represented by nine STs, all of which were restricted to serotype 23F.
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      Six isolates were related to the highly successful Taiwan19F-ST236 (PMEN14) lineage, including ST320 (19A). In the USA, the ST320 complex was responsible for a dramatic increase in 19A IPD. 19A/ST320 and related 19A strains became the most commonly recovered strains from IPD during the 2006–2009 post-PCV7 era, and also constituted the majority of multi-resistant strains during this period.
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      Putative vaccine escape ST695 19A variants (n = 2) of the non-resistant major PCV7 type 4 strain were also identified among the study isolates in the post-PCV7 period.
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      Isolates belonging to other PMEN included serotype 15B and 19A capsular variant of Netherlands15B-ST199 (PMEN37), serotype 7F PMEN39 (Netherlands7F-ST191), serotype 4 PMEN38 (Sweden4-ST205), and five ST180 serotype 3 isolates belonging to Netherlands3-ST180 (PMEN31). ST180 was isolated mainly from adults with IPD and was present during the pre-vaccination era and continued to circulate in 2013. One isolate from the pre-PCV7 period was recovered from a postmortem case in 1998. This ST180 clone is the major genotype of serotype 3 isolates globally, but independent lineages among type 3s in this study were also observed. In Europe and North America, there have been reports of the ST180 clone associated with invasive diseases and increased mortality.
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      J Clin Microbiol. 2006; 44: 999-1017
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      ST1262 (15C), which was first observed in the USA study,
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      was also seen in the early post-PCV phase in the present study. The STs associated with 6B (138, 145, 146, 497, and 4241) were also different from those detected in Latin American countries.
      • Zemlickova H.
      • Crisostomo I.
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      • Camou T.
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      Some level of marginal shift in the serotype distribution appeared to be present in this study, including the emergence of the NVTs that have been observed globally. While data suggest the presence of a diverse array of genotypes, some well described global clones and their capsular variants described in other regions of the world, in particular North America, and mainly the USA, are also circulating in Trinidad and Tobago. The STs were slightly different from those in the neighboring Latin American country Venezuela, probably because the Venezuelan isolates have been less studied. This information will continue to provide baseline data for additional studies needed on the molecular characterization of pneumococcal isolates, not only in Trinidad and Tobago, but also throughout the Caribbean region.
      One of the limitations of this study was the relatively small number of isolates used in the analysis, particularly for the pre-vaccination period (1997 to February 2010), which may not have reflected the true picture of circulating pneumococcal serotypes in Trinidad and Tobago. The isolates were recovered from patients seen and treated at the regional hospitals and this may have led to a bias towards the more severe cases. Despite these limitations, the information obtained from this study provides the first insight into the prevailing genotypes of pneumococcal isolates in the country. The findings also suggest that PCV13 could provide greater serotype coverage than PCV10 for serotypes currently causing IPD in Trinidad and Tobago. A more detailed population study is required post-PCV13 introduction in order to obtain a comprehensive picture of the circulating pneumococcal genotypes in the country.

      Acknowledgements

      MNL and PEA particularly wish to thank all the Medical Laboratory technologists and staff at the several hospitals who assisted in collecting the isolates for this study, as well as the Caribbean Public Health Agency (CARPHA) for some of the historical pneumococcal isolates. We also thank the UWI, St. Augustine Campus, for financial assistance. This publication made use of the Streptococcus pneumoniae MLST website (http://pubmlst.org/spneumoniae/) cited at the University of Oxford (Jolley and Maiden, BMC Bioinformatics, 2010;11:595). The development of this site has been funded by the Wellcome Trust.
      Conflict of interest: None declared by the authors.

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      Article info

      Publication history

      Published online: April 07, 2016
      Accepted: April 3, 2016
      Received in revised form: March 22, 2016
      Received: December 15, 2015
      Corresponding Editor: Eskild Petersen, Aarhus, Denmark.

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      DOI: https://doi.org/10.1016/j.ijid.2016.04.005

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      © 2016 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.

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