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      Detection of Streptococcus pneumoniae from Different Types of Nasopharyngeal Swabs in Children

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          Abstract

          Background

          A better understanding of the epidemiology of nasopharyngeal carriage of Streptococcus pneumoniae is important to assess the impact of vaccination and the pathogenesis of pneumococcal disease. We compared the recovery of S. pneumoniae from nylon flocked, Dacron and rayon swabs.

          Methods

          The recovery of S. pneumoniae from mocked specimens using flocked, Dacron and rayon swabs were compared by culture. The yield from paired nasopharyngeal (NP) samples obtained from healthy children sampled with flocked and Dacron swabs was also determined using culture and lytA-targeted real-time polymerase chain reaction (qPCR).

          Results

          Using mock specimen, the percentage recovery of S. pneumoniae ATCC 49619 (serotype 19F) strain from the flocked swabs was 100%, while it was 41% from Dacron swabs and 7% from rayon swabs. Similar results were observed for S. pneumoniae serotypes 1 and 5. S. pneumoniae was cultured from 18 of 42 (43%) paired NP samples from the healthy children (median age 8 [interquartile range (IQR) 5–16] months). The median number of colony-forming units (CFU) recovered from flocked swabs was two-fold higher (8.8×10 4 CFU/mL [IQR, 2.0×10 2 – 4.0×10 5 CFU/mL]) than Dacron swabs (3.7×10 4 CFU/mL [IQR, 4.0×10 2–3.2×10 5 CFU/mL], p = 0.17). Using lytA-targeted qPCR from paired NP samples, the median copy number of S. pneumoniae detected from flocked swabs was significantly higher than from Dacron swabs (3.0×10 5 genome copies/mL [IQR, 1.3×10 2−1.8×10 6] vs. 9.3×10 4 genome copies/mL [IQR, 7.0×10 1−1.1×10 6]; p = 0.005).

          Conclusion

          Flocked swabs released more S. pneumoniae compared to both Dacron and rayon swabs from mock specimens. Similarly, higher bacterial loads were detected by qPCR from flocked swabs compared with Dacron swabs from healthy children.

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          Most cited references32

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          Epidemiology and etiology of childhood pneumonia.

          Childhood pneumonia is the leading single cause of mortality in children aged less than 5 years. The incidence in this age group is estimated to be 0.29 episodes per child-year in developing and 0.05 episodes per child-year in developed countries. This translates into about 156 million new episodes each year worldwide, of which 151 million episodes are in the developing world. Most cases occur in India (43 million), China (21 million) and Pakistan (10 million), with additional high numbers in Bangladesh, Indonesia and Nigeria (6 million each). Of all community cases, 7-13% are severe enough to be life-threatening and require hospitalization. Substantial evidence revealed that the leading risk factors contributing to pneumonia incidence are lack of exclusive breastfeeding, undernutrition, indoor air pollution, low birth weight, crowding and lack of measles immunization. Pneumonia is responsible for about 19% of all deaths in children aged less than 5 years, of which more than 70% take place in sub-Saharan Africa and south-east Asia. Although based on limited available evidence, recent studies have identified Streptococcus pneumoniae, Haemophilus influenzae and respiratory syncytial virus as the main pathogens associated with childhood pneumonia.
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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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              Which pneumococcal serogroups cause the most invasive disease: implications for conjugate vaccine formulation and use, part I.

              We analyzed >70 recent data sets to compare the serogroups causing invasive pneumococcal disease (IPD) with those represented in conjugate vaccine formulations. Five to 8 and 10-11 serogroups comprise at least 75% of pneumococcal isolates from young children and older children/adults, respectively, in each geographic region. Serogroups in the 7-valent formulation (4, 6, 9, 14, 18, 19, and 23) cause 70%-88% of IPD in young children in the United States and Canada, Oceania, Africa, and Europe, and 6% of IPD in each region, including Europe, except the United States and Canada and Oceania. In contrast, several serogroups not found in 7-, 9-, and 11-valent conjugate formulations are significant causes of disease in older children/adults. Nevertheless, each conjugate formulation could prevent a substantial IPD burden in each region and age group.
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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
                2013
                26 June 2013
                : 8
                : 6
                : e68097
                Affiliations
                [1 ]Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
                [2 ]Institute for Infectious Diseases and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
                [3 ]Academic Department of Paediatrics, Wright–Fleming Institute, Imperial College London, St Mary's Campus, London, United Kingdom
                [4 ]Department of Paediatrics and Child Health, University of Cape Town, South Africa
                [5 ]Red Cross War Memorial Children's Hospital, Cape Town, South Africa
                [6 ]National Health Laboratory Service, Groote Schuur Hospital, Cape Town, South Africa
                Centers for Disease Control & Prevention, United States of America
                Author notes

                Competing Interests: Prof. Heather J. Zar and Prof. Mark P. Nicol are members of the PLOS ONE Editorial Board. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: FD MN MK. Performed the experiments: FD EW. Analyzed the data: FD MK MN HZ EW. Contributed reagents/materials/analysis tools: MN HZ. Wrote the paper: FD MK EW HZ MN.

                Article
                PONE-D-13-08248
                10.1371/journal.pone.0068097
                3694050
                23840817
                2a18cae8-164c-4154-b307-395a05542612
                Copyright @ 2013

                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
                : 22 February 2013
                : 24 May 2013
                Page count
                Pages: 6
                Funding
                This work was funded in part by grants from the National Institutes of Health, USA (1R01HD058971-01), the Medical Research Council of South Africa, the Wellcome Trust and the Bill and Mellinda Gates Foundation Global Health Grant (OPP1017641). Felix S. Dube is supported by the National Research Foundation of South Africa; Mamadou Kaba is a recipient of Carnegie Corporation of New York (USA) fellowship; Elizabeth Whittaker is supported by the Wellcome Trust, UK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Microbiology
                Bacterial Pathogens
                Streptococci
                Molecular Cell Biology
                Population Biology
                Epidemiology
                Infectious Disease Epidemiology
                Medicine
                Clinical Research Design
                Epidemiology
                Diagnostic Medicine
                Epidemiology
                Infectious Disease Epidemiology
                Pediatric Epidemiology
                Infectious Diseases
                Bacterial Diseases
                Bacterial Pneumonia
                Pneumococcus
                Streptococcal Infections

                Uncategorized
                Uncategorized

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