Document Type
Theses, Ph.D
Rights
This item is available under a Creative Commons License for non-commercial use only
Abstract
Human papillomaviruses and Chlamydia trachomatis are two of the most common sexually transmitted infections worldwide. They can be detected using urine, swab and cervical scrapings from the anogenital region. Liquid-based cervical cytology is fast becoming the method of choice for the preparation of cervical smears, with the major advantage of extra cellular material from which nucleic acids can be extracted and molecular tests performed. In this project, nucleic acid based methods for the detection of HPV and C. trachomatis and the quantitation and typing of HPV from PreservCyt cervical material were established and applied in an epidemiological study of the prevalence of these organisms in the female Irish population. The study was divided into three parts A: -Optimisation of nucleic acid extraction from PreservCyt cervical samples, B:- Development of multiplex PCR for the simultaneous detection of HPV and C. trachomatis and the determination of the prevalence of these organisms and C:- HPV genotyping, quantitation and evaluation of the relationship of these correlates with cervical neoplasia and patient factors including age and smoking status. In the first paper published from this study, three methods for DNA extraction from PreservCyt cervical samples were compared by the downstream PCR amplification of C. trachomatis using the CTP plasmid (201 bp) and MOMP gener (540 bp) primers (Keegan et al, 2005a). C. trachomatis bacterial load was calculated by real-time LightCycler PCR for the amplification of the hsp60 gene (650 bp). The Proteinase K-chelex digestion method and QIAamp method liberated similar bacterial copy numbers however the commercial Q IAamp DNA extraction kit was the most efficient methods for the preparation of DNA for PCR amplification regardless of amplicon size. The second paper published from the study details optimization and evaluation of a multiplex PCR for the simultaneous detection of HPV and C. trachomatis from PreservCyt cervical samples (Keegan et, 2005b). This multiplex was then applied to a cohort of 997 PreservCyt cervical samples from Irish women undergoing opportunistic cervical screening. The prevalence of HPV and C. trachomatis were 20% and 5% respectively. Prevalence was highest for both organisms in the under 25 years age group and decreased with age (P>0.0001). A coinfection rate of 1% was established fro HPV and C. trachomatis. HPV was associated with abnormal cytological smear results and HPV detection was 100% sensitive for the detection of high-grade cervical intraepithelial lesions. C. Trachomatis infection was not associated with abnormal cytology. In the final part of this study (paper in preparation), HPV infections were investigated further in terms of viral load and genotype. HPV viral load was determined by real time quantitative LightCycler PCR. Viral load was higher in women with borderline cytology or CIN lesions than in women with normal smears (P<0.001), however viral load did not increase with with grade of CIN. Genotyping was performed by sequencing of the MY09/11 L1 gene PCR product. High-risk HPV types predominated in all grades of cytology including normal smears. HPV16 and 18 were the most common followed by high-risk types 66, 63 and 33 and low-risk types 6, 61 and 70. HPV16 and 18 were the most common in all grades of cytology except in borderline cases where HPV66 was the most common. Cigarette smoking was more common in women with abnormal cytology (P<0.001) and also in women with evidence of HPV (P=0.008) or C. trachomatis infection (P=0.001). The main findings of this study were the overall prevalence of HPV and C. trachomatis in an opportunistically screened Irish female population; the strong association between HPV infection and high grade premalignant neoplasis; that HPV viral load was not significantly different in HPV positive cases with mild and high-grade cervical abnormalities: the commonest high-risk HPV types were 16, 18, 66, 53 and 33 and low risk types 6, 61 and 70. Cigarette smoking was strongly associated with abnormal cytology smear results and HPV, C. trachomatis and abnormal cytology smear results were more common in women under 35 years of age. The results of this study are similar to findings in other European countries. Human papillomaviruses and Chlamydia trachomatis are two of the most common sexually transmitted infections worldwide. They can be detected using urine, swab and cervical scrapings from the anogenital region. Liquid-based cervical cytology is fast becoming the method of choice for the preparation of cervical smears, with the major advantage of extra cellular material from which nucleic acids can be extracted and molecular tests performed. In this project, nucleic acid based methods for the detection of HPV and C. trachomatis and the quantitation and typing of HPV from PreservCyt cervical material were established and applied in an epidemiological study of the prevalence of these organisms in the female Irish population. The study was divided into three parts A: -Optimisation of nucleic acid extraction from PreservCyt cervical samples, B:- Development of multiplex PCR for the simultaneous detection of HPV and C. trachomatis and the determination of the prevalence of these organisms and C:- HPV genotyping, quantitation and evaluation of the relationship of these correlates with cervical neoplasia and patient factors including age and smoking status. In the first paper published from this study, three methods for DNA extraction from PreservCyt cervical samples were compared by the downstream PCR amplification of C. trachomatis using the CTP plasmid (201 bp) and MOMP gener (540 bp) primers (Keegan et al, 2005a). C. trachomatis bacterial load was calculated by real-time LightCycler PCR for the amplification of the hsp60 gene (650 bp). The Proteinase K-chelex digestion method and QIAamp method liberated similar bacterial copy numbers however the commercial Q IAamp DNA extraction kit was the most efficient methods for the preparation of DNA for PCR amplification regardless of amplicon size. The second paper published from the study details optimization and evaluation of a multiplex PCR for the simultaneous detection of HPV and C. trachomatis from PreservCyt cervical samples (Keegan et, 2005b). This multiplex was then applied to a cohort of 997 PreservCyt cervical samples from Irish women undergoing opportunistic cervical screening. The prevalence of HPV and C. trachomatis were 20% and 5% respectively. Prevalence was highest for both organisms in the under 25 years age group and decreased with age (P>0.0001). A coinfection rate of 1% was established fro HPV and C. trachomatis. HPV was associated with abnormal cytological smear results and HPV detection was 100% sensitive for the detection of high-grade cervical intraepithelial lesions. C. Trachomatis infection was not associated with abnormal cytology. In the final part of this study (paper in preparation), HPV infections were investigated further in terms of viral load and genotype. HPV viral load was determined by real time quantitative LightCycler PCR. Viral load was higher in women with borderline cytology or CIN lesions than in women with normal smears (P<0.001), however viral load did not increase with with grade of CIN. Genotyping was performed by sequencing of the MY09/11 L1 gene PCR product. High-risk HPV types predominated in all grades of cytology including normal smears. HPV16 and 18 were the most common followed by high-risk types 66, 63 and 33 and low-risk types 6, 61 and 70. HPV16 and 18 were the most common in all grades of cytology except in borderline cases where HPV66 was the most common. Cigarette smoking was more common in women with abnormal cytology (P<0.001) and also in women with evidence of HPV (P=0.008) or C. trachomatis infection (P=0.001). The main findings of this study were the overall prevalence of HPV and C. trachomatis in an opportunistically screened Irish female population; the strong association between HPV infection and high grade premalignant neoplasis; that HPV viral load was not significantly different in HPV positive cases with mild and high-grade cervical abnormalities: the commonest high-risk HPV types were 16, 18, 66, 53 and 33 and low risk types 6, 61 and 70. Cigarette smoking was strongly associated with abnormal cytology smear results and HPV, C. trachomatis and abnormal cytology smear results were more common in women under 35 years of age. The results of this study are similar to findings in other European countries.
Recommended Citation
Successfully submitted for the award of Doctor of Philosophy (Ph.D) to the Dublin Institute of Technology
Publication Details
Successfully submitted for the award of Doctor of Philosophy (Ph.D) to the Dublin Institute of Technology 2005.