Evaluating the Sensitivity and Specificity of Dried Blood Spots by Real-Time PCR for the Common Protein Gene, lytA, of Streptococcus pneumoniae in Diagnosing Pneumococcal Disease Open Access

Steyn, Sanet (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/00000036c?locale=en
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Abstract

Acute Respiratory Infection (ARI), acute infection of the upper and/or lower respiratory tract, is the leading cause of infectious death worldwide. Approximately 70% of ARI cases are in developing countries (6), which do not have the facilities and resources for proper and potentially life saving diagnosis. Pneumonia is the most severe form of ARI and is the leading cause of death in children under the age of five worldwide, killing an approximated 1.6 million children every year (11). The most common cause of bacterial pneumonia is Streptococcus pneumoniae (74).

Diagnosis of pneumonia is a considerable obstacle to proper treatment, epidemiological studies, and estimation of disease burden (6, 87, 86, 72); it is especially difficult in developing countries where pneumonia is most common because of the basic problem of acquiring a specimen for culture and analysis. Preliminary diagnosis in children is done ideally in developed countries by chest X-ray followed if possible, by further molecular testing (49). Real-time PCR may currently be the best method for identifying the microbial cause of pneumonia from a blood specimen (72), but the facilities for any of these tests are often not available in rural areas of developing countries.

Dried blood spots (DBS) have shown success in studies diagnosing HIV infection in infants safely and with ease (83,75). Whole blood can be spotted directly onto filter cards, which are chemically activated to bind DNA and inactivate pathogens (63). DBS are easier to collect, transport, store, and safer to handle than liquid samples (5).

This study proposes to use DBS to collect blood samples from patients with severe acute respiratory infection (SARI) in South Africa and ship them to the facilities at CDC to be tested for the presence of the common pneumococcal protein gene, lytA. By comparing two kinds of filter paper and two extraction techniques it was found that the Neonatal screening cards are more sensitive to detecting Streptococcus pneumoniae infected patients. The use of DBS will provide a simple and effective method of sample collection and preservation that can be used in developing and resource poor countries for improved diagnosis of Streptococcus pneumoniae.

Table of Contents

Table Of Contents
Chapter 1. Streptococcus pneumoniae...13

1.1. Background...14
1.2. Carriage and infection...15
1.3. Pathobiology...18

I. Otitis media, sinusitis, and meningitis...18
II. Pneumonia...19

1.4. Identification and diagnosis of pneumococcal disease...21
1.5. Current needs in pneumococcal disease research...23

Chapter 2. Molecular Diagnosis...24

2.1. Pneumococcal genetics, the lytA gene...25
2.2. Real-time PCR...25

I. Fluorescence probes...26

2.3. Dried blood spots...27

Chapter 3. Evaluating Dried Blood Spots by RealTime PCR for lytA in Diagnosing Pneumococcal Disease...28

3.1. Purpose/ objectives...29
3.2. Description...29
3.3. Impact...30
3.4. Participating Sites...30

I. IRB approval/ exemption...31

Chapter 4. South Africa: RMPRU, WITS, and CHB...32

4.1. Study purpose and participant selection...33
4.2. Materials and methods...34

I. Sample selection...34
II. Specimen collection, transport, and storage...37
III. DNA extraction...38
IV. Quantitative real-time PCR...38
V. Standard Curve...40
VI. Collection of data...41

Chapter 5. Materials and Methods...42

5.1. Preparation of Protocols...43

I. Culturing bacteria and spiking filter cards...43
II. Removing discs from filter cards...44
III. Acquisition of spiked samples...44
IV. Determining lower limits of detection (LLD)...45

Spiked filters

V. Optimizing FTA purification reagent extraction protocol...47
VI. Optimizing Qiagen QIAamp DNA Mini Kit extraction protocol...48

5.2. Clinical Samples...51

Flow chart of methods

I. Acquisition and storage of samples...52

5.3. DNA extraction...52

I. FTA purification reagent protocol...52
II. QIAGEN DNA mini kit protocol...53

5.4. Quantitative real-time PCR...54
5.6. Collection of data and statistical analysis...57

Percent match
Chi-square test
Sensitivity and Specificity

Chapter 6. Results...59

6.1. Preparation of Protocols...60

I. Colony counts and lower limits of detection...60

6.2. Clinical dried blood spots...61

I. CDC lytA Neonatal DBS and RMPRU lytA whole blood qPCR...62

A. Statistical analyses...70
B. Bacterial load...71

6.3 Secondary comparisons of results with FTA DBS...72

I. CDC lytA FTA DBS and CDC lytA Neonatal DBS qPCR...72
II. CDC lytA FTA DBS and RMPRU lytA whole blood qPCR...72

A. Statistical analyses...81

Chapter 7. Discussion...83
References...88


APPENDIX...100

A. Abbreviations...101
B. IRB and ethics...104

I. Emory University...104
II. Centers for Disease Control and Prevention...105
III. South Africa, University of the Witwatersrand...108

C. Correlation of results with bacterial load...109
D. Results tables for secondary FTA protocol testing...118

I. CDC neonatal and CDC FTA...118


Table of Figures and Tables

FIGURES

Figure 1. QIAcard FTA filters...27

Figure 2. RMPRU blood selection, 2009...35

Figure 3. RMPRU blood selection, 2010...36

Figure 4. Neonatal and FTA DBS...37

Figure 5. Flow chart of methods...51

Figure 6. Chi-square...57

Figure 7. Chi-square results from Neonatal DBS comparison with RMPRU results...71

Figure 8. Chi-square results from FTA DBS comparison with RMPRU results...82


TABLES

Table 1. Oligonucleotide sequence of lytA primers and probe...39

Table 2. RMPRU Gene Expression Master Mix kit setup...39

Table 3. Parameters for RMPRU real-time PCR cycling conditions...40

Table 4. Specifications of the FTA protocol preparation trials...47

Table 5. Specifications of the Mini Kit protocol preparation trials...49

Table 6. Oligonucleotide sequences of the lytA and RNAse P primers and probes...55

Table 7.CDC Universal Master Mix kit setup...55

Table 8. Parameters for CDC real-time PCR cycling conditions...56

Table 9. Colony counts at differing dilutions of S. pneumoniae...60

Table 10. Real-time PCR results from the RMPRU and the CDC...61

Table 11. Neonatal DBS matched with RMPRU results...62

Table 12. FTA DBS matched with RMPRU results...72

Table 13. Appendix C. Neonatal & RMPRU results including bacterial load data...107

Table 14. Appendix D. Neonatal DBS matched with FTA DBS results...116

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