DETECTION AND QUANTIFICATION OF STREPTOCOCCUS PNEUMONIAE SEROTYPES IN THE NASOPHARYNX OF HEALTHY CHILDREN IN PERU. Open Access

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

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Rollins School of Public Health
Department	Epidemiology
Degree	MPH
Submission	Master's Thesis
Language	English
Research Field	Biology, Microbiology Health Sciences, Epidemiology
Keyword	Children Streptococcus pneumoniae Real-time PCR Quantative PCR Quantification Healthy Children Multiple Serotypes Multiplex PCR Polymerase Chain Reaction Specificity PCR S. pneumoniae Carriage Peru Serotypes Latin America Carriage Serotypes Nasopharynx Sensitivity
Committee Chair / Thesis Advisor	Klugman, Keith P, Emory University
Committee Members	Vidal Graniel, Jorge Eugenio, Emory University
Partnering Agencies	Emory University schools, faculty or affiliated programs Non-US governmental agency (e.g., Minister of Health in Haiti) University, college or educational institution (other than Emory)

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