DETECTION AND QUANTIFICATION OF STREPTOCOCCUS PNEUMONIAE SEROTYPES IN THE NASOPHARYNX OF HEALTHY CHILDREN IN PERU. Open Access
Puerini, Raymond Anthony (2011)
Published
Abstract
Background
S. pneumoniae colonizes the nasopharynx of healthy children
during the first months of
life. From previous studies using time-consuming culture - based
methodologies,
multiple S. pneumoniae serotypes were detected in ∼15%
of healthy children in
developed countries. However, improved methodologies to quantify
all possible
serotypes are needed. Studies within this thesis standardized
quantitative PCR (qPCR)
assays to quantify the most prevalent serotypes carried by Peruvian
children in order to
improve detection of all possible serotypes associated with
carriage in an individual
child.
Methods
Our parent project collected nasopharyngeal (NP) swabs (N=500) from
Andean children
<5 years of age in Cajamarca, Peru. Isolate serotypes were
identified using multiplex
PCR by conventional culture. 149 NP samples containing the most
prevalent serotypes
were then chosen and DNA was extracted from the nasopharyngeal
swab. Serotype load
(CFU/ml) and total S. pneumoniae load ( lytA DNA)
were quantified from these swabs
using qPCR. Multiple serotype prediction was based upon numeric
differences between
total load and serotype load. Samples predicted to contain multiple
serotypes (n=15) and
42 controls (predicted to contain one serotype) were further
analyzed by multiplex PCR
to identify all possible serotypes.
Results
The most prevalent serotypes detected were 6A/B, 23F, 15B/C, 19F,
19A, 9V/A. Samples
having at least a 66% difference between total load and serotype
load were more likely to
contain multiple serotypes (60.0% vs. 21.4%, respectively,
p=0.0058). Samples with less
than 106 CFU/ml difference were less likely to contain multiple
serotypes compared to
samples having more than 106 CFU/ml difference (23% vs. 50%,
respectively, p=0.0421).
Neither sample serotype nor total bacterial load were associated
with multiple serotype
carriage. Finally, the cut-point of 20% difference between
lytA and serotype-specific
DNA amount offered the best combination of sensitivity (0.78) and
specificity (0.69) for
predicting multiple serotypes.
Conclusion
A difference of > 66% between serotype load and lytA DNA
was associated with an
increase in frequency of detection of multiple carriage. This study
represents a first step
towards developing quantification assays of S. pneumoniae
serotypes carried by healthy
children. By identifying all possible serotypes within NP samples,
more effective vaccine
strategies can ultimately be formulated.
Table of Contents
Background .. 1
Methods .. 11
Results .. 23
Discussion .. 39
References .. 54
Figures .. 62
Tables .. 66
Appendices .. 88
About this Master's Thesis
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DETECTION AND QUANTIFICATION OF STREPTOCOCCUS PNEUMONIAE SEROTYPES IN THE NASOPHARYNX OF HEALTHY CHILDREN IN PERU. () | 2018-08-28 10:09:48 -0400 |
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Supplemental Files
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All Tables_Appendix C_Fig3 21apr11_FINAL.xls () | 2018-08-28 10:10:04 -0400 |
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thesis_SAS_21apr11.sas () | 2018-08-28 10:10:38 -0400 |
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