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      Population Snapshot of Streptococcus pneumoniae Causing Invasive Disease in South Africa Prior to Introduction of Pneumococcal Conjugate Vaccines

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          Abstract

          We determined the sequence types of isolates that caused invasive pneumococcal disease (IPD) prior to routine use of pneumococcal conjugate vaccines (PCV) in South Africa. PCV-13 serotypes and 6C isolates collected in 2007 (1 461/2 437, 60%) from patients of all ages as part of on-going, national, laboratory-based surveillance for IPD, were selected for genetic characterization. In addition, all 134 non-PCV isolates from children <2 years were selected for characterization. Sequence type diversity by serotype and age category (children <5 years vs. individuals ≥5 years) was assessed for PCV serotypes using Simpson’s index of diversity. Similar genotypes circulated among isolates from children and adults and the majority of serotypes were heterogeneous. While globally disseminated clones were common among some serotypes (e.g., serotype 1 [clonal complex (CC) 217, 98% of all serotype 1] and 14 [CC230, 43%)]), some were represented mainly by clonal complexes rarely reported elsewhere (e.g., serotype 3 [CC458, 60%] and 19A [CC2062, 83%]). In children <2 years, serotype 15B and 8 were the most common serotypes among non-PCV isolates (16% [22/134] and 15% [20/134] isolates, respectively). Sequence type 7052 and 53 were most common among serotypes 15B and 8 isolates and accounted for 58% (7/12) and 64% (9/14) of the isolates, respectively. Serotype 19F, 14, 19A and 15B had the highest proportions of penicillin non-susceptible isolates. Genotypes rarely reported in other parts of the world but common among some of our serotypes highlight the importance of our data as these genotypes may emerge post PCV introduction.

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          eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data.

          The introduction of multilocus sequence typing (MLST) for the precise characterization of isolates of bacterial pathogens has had a marked impact on both routine epidemiological surveillance and microbial population biology. In both fields, a key prerequisite for exploiting this resource is the ability to discern the relatedness and patterns of evolutionary descent among isolates with similar genotypes. Traditional clustering techniques, such as dendrograms, provide a very poor representation of recent evolutionary events, as they attempt to reconstruct relationships in the absence of a realistic model of the way in which bacterial clones emerge and diversify to form clonal complexes. An increasingly popular approach, called BURST, has been used as an alternative, but present implementations are unable to cope with very large data sets and offer crude graphical outputs. Here we present a new implementation of this algorithm, eBURST, which divides an MLST data set of any size into groups of related isolates and clonal complexes, predicts the founding (ancestral) genotype of each clonal complex, and computes the bootstrap support for the assignment. The most parsimonious patterns of descent of all isolates in each clonal complex from the predicted founder(s) are then displayed. The advantages of eBURST for exploring patterns of evolutionary descent are demonstrated with a number of examples, including the simple Spain(23F)-1 clonal complex of Streptococcus pneumoniae, "population snapshots" of the entire S. pneumoniae and Staphylococcus aureus MLST databases, and the more complicated clonal complexes observed for Campylobacter jejuni and Neisseria meningitidis.
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            Sustained reductions in invasive pneumococcal disease in the era of conjugate vaccine.

            Changes in invasive pneumococcal disease (IPD) incidence were evaluated after 7 years of 7-valent pneumococcal conjugate vaccine (PCV7) use in US children. Laboratory-confirmed IPD cases were identified during 1998-2007 by 8 active population-based surveillance sites. We compared overall, age group-specific, syndrome-specific, and serotype group-specific IPD incidence in 2007 with that in 1998-1999 (before PCV7) and assessed potential serotype coverage of new conjugate vaccine formulations. Overall and PCV7-type IPD incidence declined by 45% (from 24.4 to 13.5 cases per 100,000 population) and 94% (from 15.5 to 1.0 cases per 100,000 population), respectively (P< .01 all age groups). The incidence of IPD caused by serotype 19A and other non-PCV7 types increased from 0.8 to 2.7 cases per 100,000 population and from 6.1 to 7.9 cases per 100,000 population, respectively (P< .01 for all age groups). The rates of meningitis and invasive pneumonia caused by non-PCV7 types increased for all age groups (P< .05), whereas the rates of primary bacteremia caused by these serotypes did not change. In 2006-2007, PCV7 types caused 2% of IPD cases, and the 6 additional serotypes included in an investigational 13-valent conjugate vaccine caused 63% of IPD cases among children <5 years-old. Dramatic reductions in IPD after PCV7 introduction in the United States remain evident 7 years later. IPD rates caused by serotype 19A and other non-PCV7 types have increased but remain low relative to decreases in PCV7-type IPD.
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              Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA.

              The accurate diagnosis of pneumococcal disease has frequently been hampered not only by the difficulties in obtaining isolates of the organism from patient specimens but also by the misidentification of pneumococcus-like viridans group streptococci (P-LVS) as Streptococcus pneumoniae. This is especially critical when the specimen comes from the respiratory tract. In this study, three novel real-time PCR assays designed for the detection of specific sequence regions of the lytA, ply, and psaA genes were developed (lytA-CDC, ply-CDC, and psaA, respectively). These assays showed high sensitivity (<10 copies for lytA-CDC and ply-CDC and an approximately twofold less sensitivity for psaA). Two additional real-time PCR assays for lytA and ply described previously for pneumococcal DNA detection were also evaluated. A panel of isolates consisting of 67 S. pneumoniae isolates (44 different serotypes and 3 nonencapsulated S. pneumoniae isolates from conjunctivitis outbreaks) and 104 nonpneumococcal isolates was used. The 67 S. pneumoniae isolates were reactive in all five assays. The new real-time detection assays targeting the lytA and psaA genes were the most specific for the detection of isolates confirmed to be S. pneumoniae, with lytA-CDC showing the greatest specificity. Both ply PCRs were positive for all isolates of S. pseudopneumoniae, along with 13 other isolates of other P-LVS isolates confirmed to be non-S. pneumoniae by DNA-DNA reassociation. Thus, the use of the ply gene for the detection of pneumococci can lead to false-positive reactions in the presence of P-LVS. The five assays were applied to 15 culture-positive cerebrospinal fluid specimens with 100% sensitivity; and serum and ear fluid specimens were also evaluated. Both the lytA-CDC and psaA assays, particularly the lytA-CDC assay, have improved specificities compared with those of currently available assays and should therefore be considered the assays of choice for the detection of pneumococcal DNA, particularly when upper respiratory P-LVS might be present in the clinical specimen.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                18 September 2014
                : 9
                : 9
                : e107666
                Affiliations
                [1 ]Centre for Respiratory Diseases and Meningitis (CRDM), National Institute for Communicable Diseases (NICD), a division of the National Health Laboratory Service, Johannesburg, South Africa
                [2 ]Respiratory and Meningeal Pathogens Research Unit, University of the Witwatersrand, Johannesburg, South Africa
                [3 ]Medical Research Council, Johannesburg, South Africa
                [4 ]Hubert Department of Global Health, Rollins School of Public Health, and Division of Infectious Diseases, School of Medicine, Emory University, Atlanta, Georgia, United States of America
                Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano, Italy
                Author notes

                Competing Interests: Anne von Gottberg and Mignon du Plessis have received research funding from Pfizer. In addition, Anne von Gottberg has received research funding from Sanofi Pasteur. Keith P. Klugman has received consultancy fees from Pfizer, Sanofi Pasteur, and Novartis. This work was funded by the National Health Laboratory Service, Centers for Disease Control and Prevention (CDC), National Centre for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention (NCHHSTP), Global AIDS Program Cooperative Agreement U62/PSO022901. (The contents are solely the responsibility of the authors and do not necessarily represent the official views of the CDC). Molecular characterization of isolates was funded by Pfizer Vaccines Research. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: AVG KPK MDP NW. Performed the experiments: KMN LDG. Analyzed the data: KMN LDG MDP. Contributed reagents/materials/analysis tools: AVG MDP. Contributed to the writing of the manuscript: KMN LDG AVG KPK MDP NW.

                Article
                PONE-D-14-19229
                10.1371/journal.pone.0107666
                4169438
                25233455
                2af3f647-05b3-4299-ac74-dfcebbf853a4
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 29 April 2014
                : 12 August 2014
                Page count
                Pages: 11
                Funding
                This work was funded by the National Health Laboratory Service, Centers for Disease Control and Prevention (CDC), National Centre for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention (NCHHSTP), Global AIDS Program Cooperative Agreement U62/PSO022901. (The contents are solely the responsibility of the authors and do not necessarily represent the official views of the CDC). Molecular characterization of isolates was funded by Pfizer Vaccines Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Streptococcus
                Pneumococcus
                Plant Science
                Plant Pathology
                Infectious Disease Epidemiology
                Medicine and Health Sciences
                Epidemiology
                Molecular Epidemiology
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

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